WO2016112271A1 - Compositions et procédés d'accélération de la résolution d'une inflammation pulmonaire aiguë - Google Patents

Compositions et procédés d'accélération de la résolution d'une inflammation pulmonaire aiguë Download PDF

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Publication number
WO2016112271A1
WO2016112271A1 PCT/US2016/012621 US2016012621W WO2016112271A1 WO 2016112271 A1 WO2016112271 A1 WO 2016112271A1 US 2016012621 W US2016012621 W US 2016012621W WO 2016112271 A1 WO2016112271 A1 WO 2016112271A1
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subject
lung injury
disorder
inflammatory disease
injury event
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PCT/US2016/012621
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English (en)
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Franco D'ALESSIO
Benjamin David SINGER
Landon KING
Neil Raj AGGARWAL
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The Johns Hopkins University
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Priority to US15/542,380 priority Critical patent/US10905706B2/en
Publication of WO2016112271A1 publication Critical patent/WO2016112271A1/fr
Priority to US17/139,665 priority patent/US20210275561A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A61K31/245—Amino benzoic acid types, e.g. procaine, novocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • This invention relates generally to mechanisms that accelerate resolution and repair of lung damage.
  • the invention relates generally to epigenetic mechanisms that enhance regulatory T cell (Tregs) to promote resolution of lung injury/inflammatory lung damage.
  • the invention also relates to the uses of IL-4 (optionally, recombinant IL-4 ("rIL-4")) as an appropriate immunotherapy for lung injury resolution.
  • Acute respiratory distress syndrome an inflammatory condition caused by direct or indirect lung injury, is a common and life-threatening disease without effective pharmacotherapy (1).
  • Mechanisms leading to resolution and repair from acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain incompletely understood.
  • alveolar macrophages acquire a Ml phenotype and secrete classical Ml predominant cytokines. Less is known about the alveolar macrophage phenotype during resolution of lung inflammation, and whether and/or how this might impact lung repair.
  • Resolution of inflammation following acute lung injury is an active process (2) that requires Foxp3+ regulatory T cells (Tregs) in a direct lung injury mouse model (intratracheal lipopolysaccharide (LPS) administration (3)).
  • the invention is based, at least in part, upon the discovery that administration of a DNA methyltransferase inhibitor, either systemically to a subject or to T cells (Treg cells) in culture for transfer to the subject, exerted a remarkable effect in promoting/enhancing recovery of the subject from lung injury.
  • the invention is also based, at least in part, upon the discovery that administration of IL-4 to a subject having a lung injury exerted a remarkable effect in promoting/enhancing recovery of the subject from lung injury.
  • the invention provides a method for treating an acute inflammatory disease or disorder in a subject that involves identifying a subject having an acute
  • a DNA methyltransferase inhibitor to the subject, thereby treating the acute inflammatory disease or disorder in the subject.
  • the invention provides a method for treating lung injury in a subject that involves identifying a subject having a lung injury, and administering a DNA
  • methyltransferase inhibitor to the subject, thereby treating the lung injury in the subject.
  • the invention provides a method for treating influenza in a subject that involves identifying a subject having influenza, and administering a DNA methyltransferase inhibitor to the subject, thereby treating influenza in the subject.
  • the DNA methyltransferase inhibitor is administered in an amount sufficient to increase Treg frequency in the subject.
  • the DNA methyltransferase inhibitor is administered in an amount sufficient to increase Treg frequency in the subject.
  • methyltransferase inhibitor is administered in an amount sufficient to increase Foxp3 expression in the Treg cells of the subject.
  • the DNA methyltransferase inhibitor is 5-Azacytidine; 5- Aza-2'-deoxycytidine; zebularine; 5-Fluoro-2'-deoxycytidine; 5, 6-Dihydro-5-azacytidine; Hydralazine; Procainamide; Procaine; EGCG ((-)-epigallocatechin-3-gallate); Psammaplin A; MG98; RG108; a DNA methyltransferase-targeting antisense oligonucleotide; or a DNA methyltransferase-targeting RNAi agent.
  • the DNA methyltransferase inhibitor is 5-Aza-2'-deoxycytidine (decitabine; DAC).
  • the acute inflammatory disease or disorder or lung injury is an acute lung injury, optionally, acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • the subject is a human.
  • the DNA methyltransferase inhibitor is administered to the subject at least 2-3 hours after a lung injury event in the subject, at least 6 hours after a lung injury event in the subject, at least 12 hours after a lung injury event in the subject, at least 24 hours after a lung injury event in the subject, at least 36 hours after a lung injury event in the subject, at least 48 hours after a lung injury event in the subject, at least 72 hours after a lung injury event in the subject, at least four days after a lung injury event in the subject, at least five days after a lung injury event in the subject, at least six days after a lung injury event in the subject, at least seven days after a lung injury event in the subject, at least eight days after a lung injury event in the subject, at least nine days after a lung injury event in the subject, or at least ten days after a lung injury event in the subject.
  • the DNA methyltransferase inhibitor is administered to the subject within 1-2 or 1-3 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 6 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 12 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 24 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 36 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 48 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 72 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within four days after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within five days after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within six days after diagnosis of an
  • An additional aspect of the invention provides a method for treating an acute inflammatory disease or disorder in a subject that involves identifying a subject having an acute inflammatory disease or disorder; obtaining a population of cells that includes T cells; administering a DNA methyltransferase inhibitor to the population of cells in vitro, thereby generating a treated population of cells; and administering the treated population of cells to the subject, thereby treating the acute inflammatory disease or disorder in the subject.
  • Another aspect of the invention provides a method for treating influenza in a subject that involves identifying a subject having influenza; obtaining a population of cells that includes T cells; administering a DNA methyltransferase inhibitor to the population of cells in vitro, thereby generating a treated population of cells; and administering the treated population of cells to the subject, thereby treating influenza in the subject.
  • the cells are autologous cells. In another embodiment, the cells are allogeneic cells.
  • the cells are isolated from the blood, spleen, thymus and/or lymph of a subject (optionally, the subject being administered the DNA methyltransferase inhibitor-treated cells, though in certain embodiments, a distinct subject than the one to which the DNA methyltransferase inhibitor-treated cells are administered).
  • the DNA methyltransferase inhibitor is administered for at least 24 hours to the population of cells in vitro.
  • Another aspect provides a DNA methyltransferase inhibitor-treated population of cells obtained by a method of the invention.
  • a further aspect of the invention provides a pharmaceutical composition for the treatment of an acute inflammatory disease or disorder or influenza that includes a DNA methyltransferase inhibitor and a pharmaceutically acceptable carrier.
  • the invention provides a method for treating an acute inflammatory disease or disorder in a subject, the method involving identifying a subject having an acute inflammatory disease or disorder, and administering an IL-4 polypeptide or fragment thereof possessing cytokine activity, or an IL-4 agonist to the subject, thereby treating the acute inflammatory disease or disorder in the subject.
  • Another aspect of the invention provides a method for treating a lung injury in a subject, the method involving identifying a subject having a lung injury, and administering an IL-4 polypeptide or fragment thereof possessing cytokine activity, or an IL-4 agonist to the subject, thereby treating the lung injury in the subject.
  • the invention provides a method for treating influenza in a subject that involves identifying a subject having influenza, and administering an IL-4 polypeptide or fragment thereof possessing cytokine activity, or an IL-4 agonist to the subject, thereby treating influenza in the subject.
  • the invention provides a method for treating a live bacterial infection in a subject that involves identifying a subject having a live bacterial infection, and administering an IL-4 polypeptide or fragment thereof possessing cytokine activity, or an IL- 4 agonist to the subject, thereby treating the live bacterial infection in the subject.
  • the live bacterial infection is Staphylococcus, Pseudomonas, Streptococcus, or a combination thereof.
  • the live bacterial infection is Staphylococcus, Pseudomonas, Streptococcus, or a combination thereof.
  • the live bacterial infection is Staphylococcus, Pseudomonas, Streptococcus, or a combination thereof.
  • the live bacterial infection is Staphylococcus, Pseudomonas, Streptococcus, or a combination thereof.
  • the live bacterial infection is
  • Staphylococcus aureus Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae or a combination thereof.
  • the IL-4 polypeptide or fragment thereof possessing cytokine activity or IL-4 agonist is a recombinant polypeptide.
  • the IL-4 polypeptide or fragment thereof possessing cytokine activity or IL-4 agonist is a recombinant IL-4 polypeptide.
  • the recombinant IL-4 polypeptide is selected from the group consisting of a recombinant murine IL-4 of SEQ ID NO: 1 or a fragment thereof possessing cytokine activity and a recombinant human IL-4 of SEQ ID NO: 3 or a fragment thereof possessing cytokine activity.
  • the acute inflammatory disease or disorder or lung injury is an acute lung injury.
  • the acute inflammatory disease or disorder or lung injury is acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • the subject is human.
  • the IL-4 polypeptide or fragment thereof possessing cytokine activity or IL-4 agonist is administered to the subject at least 2-3 hours after a lung injury event in the subject, at least 6 hours after a lung injury event in the subject, at least 12 hours after a lung injury event in the subject, at least 24 hours after a lung injury event in the subject, at least 36 hours after a lung injury event in the subject, at least 48 hours after a lung injury event in the subject, at least 72 hours after a lung injury event in the subject, at least four days after a lung injury event in the subject, at least five days after a lung injury event in the subject, at least six days after a lung injury event in the subject, at least seven days after a lung injury event in the subject, at least eight days after a lung injury event in the subject, at least nine days after a lung injury event in the subject, or at least ten days after a lung injury event in the subject.
  • the IL-4 polypeptide or fragment thereof possessing cytokine activity or IL-4 agonist is administered to the subject within 1-2 or 1-3 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 6 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 12 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 24 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 36 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 48 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within 72 hours after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within four days after diagnosis of an acute inflammatory disease or disorder, a lung injury event or influenza in the subject, within five days after diagnosis of an acute inflammatory disease or disorder, a lung injury event
  • An additional aspect of the invention provides a pharmaceutical composition for the treatment of an acute inflammatory disease or disorder that includes an IL-4 polypeptide or fragment thereof possessing cytokine activity or IL-4 agonist and a pharmaceutically acceptable carrier.
  • FIGS 1A to ID show that DAC increased lung Treg frequency and Foxp3 expression under sham injury conditions.
  • Lung CD4+ CD25+ Foxp3+ cells are shown in wild type (WT) mice as a frequency of total lung cells five days after receiving intratracheal (i.t.) water.
  • Lung CD4+CD25 hl Foxp3+ cells are shown in wild type (WT) mice as number in the right lung, frequency of lung cells, and frequency of CD4+ cells five days after receiving intratracheal (i.t.) water.
  • WT wild type mice
  • Foxp3 expression was determined by fluorescence in lung Tregs five days after i.t. water; values reported are mean fluorescence intensities + SEM.
  • Figures 2 A to 2E demonstrate that DAC treatment promoted resolution of lung injury in WT mice.
  • body weight relative to baseline was plotted after injury.
  • BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in WT mice two and five days after injury with intratracheal (i.t.) LPS.
  • Figure 2E lung sections two and five days after injury were stained with H&E.
  • Original magnification: x20; x200 (insets), n 8 per group; * P ⁇ 0.05, ⁇ P ⁇ 0.001, Mann- Whitney U test. Values reported are mean + SEM.
  • Figures 3A to 3D demonstrate that lung Treg frequency, activation state, suppressive phenotype, and proliferative capacity increased with DAC treatment after injury.
  • lung CD4 + CD25 hl Foxp3 + cells are shown within a fixed sample of cells from the right lung (10 5 cells) two and five days post-injury in WT mice; in Figure 3 A, middle panel, lung CD4 + CD25 hi Foxp3 + cells are shown as a frequency of total lung cells two and five days post-injury in WT mice; and in Figure 3A, right panel, lung CD4 + CD25 hl Foxp3 + cells are shown as a percentage of total CD4 + cells two and five days post-injury in WT mice.
  • FIGs 4 A to 4E show that lymphocyte-deficient (Rag- ⁇ 7 ) mice did not resolve lung injury in response to DAC treatment.
  • body weight relative to baseline was plotted after injury.
  • BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in Rag-1-/- mice five days after injury with LPS.
  • lung sections were stained with H&E.
  • Original magnification: x20; x200 (insets), n 5 per group; P > 0.05, Mann- Whitney U test. Values reported are mean + SEM.
  • Figures 5 A to 5E show that DAC did not promote resolution of lung injury in Treg-depleted mice.
  • body weight relative to baseline was plotted beginning with the first diphtheria toxin dose (two days before injury). Arrowheads represent diphtheria toxin (DTx) doses.
  • BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in diphtheria toxin-treated Foxp3DTR mice (Foxp3DTR DTx+) five days after injury with LPS.
  • lung sections were stained with H&E.
  • FIGS 6A to 6E demonstrate that DAC altered CD4+ T cell phenotype and function in vitro.
  • splenic WT CD4 + CD25 " conventional T cells (Tconv) or Tregs were cultured with indicated amounts of DAC for 48 hours. Foxp3 fluorescence was plotted for indicated DAC concentrations (vehicle/0, 10, and 100 nM).
  • Figures 6B and 6C demonstrate that DAC altered CD4+ T cell phenotype and function in vitro.
  • CD4+CD25 hi Foxp3+ Treg expression of CD44, CD39, and CTLA-4 B and the percentage of total Ki-67+ Tregs (C) were determined byfluorescence in cultured Tregs treated with 100 nM DAC or vehicle.
  • Accompanying bar graphs show summary mean fluorescence intensities ( Figures 6A and 6B) and the percentage of Ki-67+ Tregs ( Figure 6C).
  • T effector cells Teff
  • Tregs were previously cultured in the presence of 100 nM DAC or vehicle.
  • FIGs 7 A to 71 demonstrate that adoptive transfer (AT) of DAC-treated Tregs rescued the injury phenotype in Treg-depleted mice (Foxp3DTR DTx+ ).
  • body weight relative to baseline was plotted after injury.
  • Arrowheads represent diphtheria toxin (DTx) doses.
  • BAL total protein (B), total cell counts (C), and neutrophil counts (D) were determined in Treg-depleted mice seven days after injury with LPS.
  • Figure 7E lung sections seven days after injury were stained with H&E.
  • FIG. 7F BAL active TGF- ⁇ was assessed in vehicle- and DAC-treated Tregs at seven days post-injury. TGF- ⁇ concentrations were measured in BAL fluid.
  • Figure 7G shows that exogenous lung Tregs (Foxp3-APC+ GFP-; ie.
  • CD4+CD25 hi APC+GFP- are shown as number in the right lung, frequency of lung cells, and frequency of CD4+ cells seven days post-injury.
  • Exogenous lung Tregs were significantly elevated in DAC-treated Treg populations, when assessed in a fixed number of cells (left panel), as a percentage of total cell count (middle panel), or as a percentage of CD4 + cells (right panel).
  • Figures 7H and 71 Foxp3 (H) and CD44, CD39, CTLA-4 expression, and the percentage of Ki-67+ Tregs (I) were determined by fluorescence in exogenous (APC+ GFP-) lung Tregs seven days after injury.
  • Figures 8A to 8G show that DAC rescue treatment had favorable effects in an influenza (flu) model.
  • Figures 8A to 8G demonstrate the significant, beneficial impact of DAC treatment upon weight (Figure 8A), BAL levels (Figures 8B to 8D) histological recovery from influenza (Figure 8E), and Treg levels ( Figures 8F and 8G) in a mouse model of influenza challenge.
  • body weight relative to baseline is shown 10 days after inoculation with influenza.
  • BAL total protein Figure 8B
  • total cell counts Figure 8C
  • neutrophil counts Figure 8D
  • FIG. 8F lung CD4+CD25 hi Foxp3+ cells are shown as number in the right lung, frequency of lung cells, and frequency of CD4+ cells 10 days post-inoculation.
  • Figure 9 shows that flow cytometry confirmed successful adoptive transfer and homing to the lung.
  • Figure 9 is an example analysis of lung single cell suspension.
  • Cells were first gated based on SSC/FSC (side scatter/forward scatter, the distribution of cells based on intracellular composition and size, respectively) to remove debris and gate on the characteristic low SSC/FSC of lymphocytes.
  • Live cells were identified using a UV-excitable dead cell dye.
  • CD4+, CD4+CD25 hi , and CD4+CD25 hi Foxp3+ cells (Tregs) were gated as shown.
  • the GFP- (exogenous) cell fraction was identified following adoptive transfer (AT); for comparison, the dashed histogram shows an undepleted Foxp3DTR mouse that did not receive adoptive transfer. Biexponential scaling was used when more than 5% of events fell on the axis.
  • Figures 10A to IOC show that DAC treatment did not affect lung CD4+Foxp3- cell frequency or phenotype.
  • Systemic DAC treatment does not affect lung CD4+Foxp3- cells after injury.
  • lung CD4 + Foxp3 " cells are shown as a frequency of lung cells five days post-injury in WT mice.
  • CD44, CTLA-4, and CD25 expression were determined by fluorescence in lung CD4 + Foxp3 " cells five days after injury.
  • Figure 11 shows that DAC treatment significantly elevated spleen Treg levels, at either five days post- LPS challenge or at five days post- water treatment.
  • Systemic DAC treatment affected splenic Treg frequency.
  • Figures 12 A to 12E show lung Treg depletion in diphtheria toxin-treated Foxp3DTR mice compared to diphtheria toxin-treated LPS-injured WT mice 5 days post-injury (Figure 12A).
  • WT mice treated with diphtheria toxin experienced accelerated lung injury resolution in response to DAC ( Figures 12B to 12E).
  • DAC promoted lung injury resolution in diphtheria toxin-treated WT mice.
  • Figure 12A shows lung CD4+CD25 hl Foxp3+ cells as number in the right lung, frequency of lung cells, and frequency of CD4+ cells five days post-injury in diphtheria toxin-treated Foxp3DTR mice (Foxp3DTR DTx+) and diphtheria toxin-treated wild type mice (WT DTx+).
  • Figures 12B to 12D shows BAL total protein (Figure 12B), total cell counts (Figure 12C), and neutrophil counts (Figure 12D) were determined in diphtheria toxin-treated WT mice five days after injury with LPS.
  • Figure 12E lung sections were stained with H&E.
  • Original magnification, x20; x200 (insets), n 5 per group; * P ⁇ 0.05, Mann-Whitney U test. Values reported are mean + SEM.
  • Figures 13A and 13B show that DAC increased lung Treg number and Foxp3 expression under sham injury conditions.
  • lung CD4+CD25 hi Foxp3+ cells are shown in wild type (WT) mice as number in the right lung, frequency of lung cells, and frequency of CD4+ cells five days after receiving intratracheal (i.t.) water.
  • Foxp3 expression was determined by fluorescence in lung Tregs five days after i.t. water.
  • the accompanying bar graph shows summary mean fluorescence intensities. * P ⁇ 0.05, ⁇ P ⁇ 0.001, Mann- Whitney U test. Values reported are mean + SEM.
  • Figures 14A to 14H show that WT (C57BL/6) mice treated with IL-4 demonstrated improved survival and accelerated ALI resolution.
  • Figure 14C shows representative H&E stain of the lung at day 6 after i.t. LPS in sham- or IL-4- treated mice at 4x and 20x (inserts) magnification.
  • Figure 14H shows dynamic lung compliance (Crs, ml/cmH20) and diffusing capacity (DFCO) after i.t. LPS or i.t.
  • Figures 16A to 16G show that lung macrophages prominently expressed M2 proteins following IL-4-treatment in i.t. LPS-exposed WT mice.
  • Figure 16B shows whole lung tissue immunoblots for Argl, Yml, FIZZ1, and ⁇ -actin after i.t. LPS or H20.
  • Figure 16E shows intracellular FIZZ1 expression quantified by the number of positive cells and MFI for monocyte (Mo,
  • Figure 16G shows the M2 markers MMR and Dectin-1, and the Ml marker CD86, quantified on the surface of F4/80+ macrophages at day 6 after i.t. LPS.
  • Figures 17 A to 17 C show that macrophage depletion mitigated IL-4 benefits on ALI resolution.
  • Figures 18A to 18E show that IL-4 did not accelerate ALI resolution in Stat6 _/ ⁇ mice.
  • Figure 18A shows weight change
  • Figure 18B shows BAL protein
  • Figure 18C shows neutrophils
  • WT wild-type
  • BAL protein * p ⁇ 0.05 compared to WT sham, Stat6 _/" IL-4 and *** p ⁇ 0.001 compared to Stat6 _/" sham by t-test.
  • Figures 19A to 19H show that regulatory T-cells (Tregs) were not necessary for IL-4 to accelerate ALI resolution.
  • Figure 19B shows the experimental design for Treg depletion in Foxp3DTR mice using i.p. injection of diphtheria toxin (DT). All Foxp3DTR mice received DT to deplete Tregs and either IL-4 or sham treatment following i.t. LPS.
  • DT diphtheria toxin
  • Figure 20A to 20E show that IL-4 accelerated ALI resolution following lung Pseudomonas challenge.
  • BAL protein Figure 20A
  • BAL albumin Figure 20B
  • BAL neutrophils Figure 20C
  • BAL macrophages Figure 20D
  • the present invention relates, at least in part, to the unexpected observation that administration of a DNA methyltransferase inhibitor or an IL-4 agent to a mammalian subject having an acute lung injury promoted recovery of the subject from the acute lung injury.
  • Administration of a DNA methyltransferase inhibitor activated and/or expanded the active population/raised the frequency of regulatory T cells in a mammalian subject having an acute lung injury, exerting a positive therapeutic outcome on recovery of the subject from the acute lung injury.
  • Treg DNMT inhibition augmented Treg suppressive phenotype and function in the subject and accelerated resolution of LPS-induced lung injury.
  • Tregs mediated the inhibitor' s pro-resolution action.
  • systemic DNMT inhibition likely acted upon multiple cell types involved in lung injury repair, Tregs were identified as appropriately depending on DNA demethylation (10) and therefore Tregs might have also been more predisposed to the effects of a DNMT inhibitor.
  • histone deacetylase inhibition can avert or mitigate lung injury (33-35), perhaps via MAP kinase pathway modulation.
  • Acute respiratory distress syndrome is a devastating inflammatory lung disease for which there are no effective targeted therapies.
  • ARDS Acute respiratory distress syndrome
  • Tregs regulatory T cells
  • Tregs themselves were highly dependent on epigenetic mechanisms, such as DNA hypomethylation.
  • Macrophages are critical for initiation of lung inflammation, but also for resolution and repair of the lung. Immunotherapy with IL-4 reprogrammed macrophages into an antiinflammatory and pro-repair phenotype.
  • rIL-4 recombinant IL-4
  • the model and findings presented herein have translational relevance to patients with ARDS or those patients at significant risk for ARDS.
  • the ability to deliver systemic immunotherapy well after onset of lung inflammation is quite appealing for a severe condition in which patients rarely seek medical care prior to the development of lung inflammation.
  • ARDS is an acute inflammatory process with a mortality in the range of 30-40%, any potential immunotherapy is promising.
  • exogenous, systemic delivery of recombinant IL-4 (rIL-4) was clearly a beneficial therapy in experimental models of lung injury, promoting resolution of acute inflammatory conditions.
  • LPS a Gram-negative bacterial cell wall component
  • the sterile inflammation model used herein is relevant to many causes of ARDS, including aspiration of gastric contents, ventilator-induced lung injury, near drowning, and collateral lung injury associated with treated infection. Additionally, it has been initially identified herein that rIL-4 can positively impact lung injury resolution following bacterial and viral pneumonia in experimental models.
  • rIL-4 has been shown to be safe and potentially effective in treating immune-mediated diseases such as psoriasis, but has never been used to treated acute inflammation as occurs in ARDS.
  • the present invention is based, at least in part, upon identification of rIL-4 as an appropriate immunotherapy for patients with ARDS, establishing a basis for clinical testing of this agent as a therapeutic.
  • DNA methyltransferase inhibitor or “DNMTi” has its general meaning in the art and refers to a lung injury treatment, e.g., systemic or ex vivo.
  • Exemplary "DNA methyltransferase inhibitors” can be sub-divided into nucleoside analogues (e.g., 5- Azacytidine (azacytidine), 5-Aza-2'-deoxycytidine (decitabine, 5-Aza-Cd; referred to as “DAC” herein), zebularine, 5-Fluoro-2'-deoxycytidine (5-F-CdR), 5, 6-Dihydro-5- azacytidine (DHAC)) and non-nucleoside analogue families (e.g., Hydralazine,
  • Regulatory T cells are also referred to as Treg and were formerly known as suppressor T cell. Regulatory T cells are a component of the immune system that suppress immune responses of other cells. Regulatory T cells usually express CD3, CD4, CD8, CD25, and Foxp3. Additional regulatory T cell populations include Thl, Th3, CD8+CD28-, CD69+, and Qa-1 restricted T cells. Regulatory T cells actively suppress activation of the immune system and prevent pathological self -reactivity, i.e. autoimmune disease. The
  • TGF-beta and Interleukin 10 have also been implicated in regulatory T cell function. Similar to other T cells, a subset of regulatory T cells can develop in the thymus and this subset is usually referred to as natural Treg (or nTreg).
  • induced Treg or iTreg can develop in the periphery from naive CD4+ T cells.
  • the large majority of Foxp3-expressing regulatory T cells are found within the major histocompatibility complex (MHC) class II restricted CD4-expressing (CD4+) helper T cell population and express high levels of the interleukin-2 receptor alpha chain (CD25).
  • MHC major histocompatibility complex
  • CD25 interleukin-2 receptor alpha chain
  • regulatory T cells do not produce IL-2 and are therefore anergic at baseline.
  • An alternative way of identifying regulatory T cells is to determine the DNA methylation pattern of a portion of the foxp3 gene (TSDR, Treg-specific-demethylated region) which is found demethylated in Tregs.
  • inflammatory disease or "inflammatory disorder” refers to pathological states resulting in inflammation, typically caused by leukocyte infiltration.
  • disorders include inflammatory skin diseases, including, without limitation, psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (IBD) (such as Crohn's disease and ulcerative colitis); ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and constriction after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma, hypovolemic shock; asphyxia; acute respiratory distress syndrome; acute-lung injury; Behcet's Disease;
  • IBD inflammatory bowel disease
  • ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and constriction after percutaneous transluminal coronary angioplasty, stroke,
  • Exemplary "acute inflammatory diseases or disorders” include, but are not limited to, acute lung injury, acute liver failure, systemic inflammatory response syndrome (SIRS), different degrees of sepsis including sepsis, severe sepsis, and septic shock, etc.
  • SIRS systemic inflammatory response syndrome
  • an effective amount includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat lung injuries.
  • An effective amount of a DNA methyltransferase inhibitor may vary according to factors such as the disease/injury state, age, and weight of the subject, and the ability of the DNA
  • an effective amount of an IL-4 agent e.g., IL-4, optionally a recombinant IL-4 or fragment thereof, or an IL-4 agonist (e.g., an activator of IL-4, an antibody, compound or other agent that activates IL- 4Ra, etc.)
  • an IL-4 agent e.g., IL-4, optionally a recombinant IL-4 or fragment thereof, or an IL-4 agonist (e.g., an activator of IL-4, an antibody, compound or other agent that activates IL- 4Ra, etc.)
  • an IL-4 agent may vary according to factors such as the disease/injury state, age, and weight of the subject, and the ability of the IL-4 agent to elicit a desired response in the subject.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of an IL-4 agent or a DNA methyltransferase inhibitor are outweighed by the therapeutically beneficial effects.
  • “Ameliorate,” “amelioration,” “improvement” or the like refers to, for example, a detectable improvement or a detectable change consistent with improvement that occurs in a subject or in at least a minority of subjects, e.g., in at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100% or in a range between any two of these values.
  • Such improvement or change may be observed in treated subjects as compared to subjects not treated with an IL-4 agent or a DNA methyltransferase inhibitor, where the untreated subjects have, or are subject to developing, the same or similar injury/condition, disease, symptom or the like.
  • Amelioration of an injury/condition, disease, symptom or assay parameter may be determined subjectively or objectively, e.g., via self- assessment by a subject(s), by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., a quality of life assessment, a slowed progression of a disease(s) or condition(s), a reduced severity of a disease(s) or condition(s), or a suitable assay(s) for the level or activity(ies) of a biomolecule(s), cell(s), by detection of respiratory or inflammatory disorders in a subject, and/or by modalities such as, but not limited to photographs, video, digital imaging and pulmonary function tests.
  • Amelioration may be transient, prolonged or permanent, or it may be variable at relevant times during or after an IL-4 agent or a DNA methy transferase inhibitor is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within timeframes described infra, or about 12 hours to 24 or 48 hours after the administration or use of an IL-4 agent or a DNA methyltransferase inhibitor to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more after a subject(s) has received such treatment.
  • the "modulation" of, e.g., a symptom, level or biological activity of a molecule, or the like refers, for example, to the symptom or activity, or the like that is detectably increased or decreased. Such increase or decrease may be observed in treated subjects as compared to subjects not treated with an IL-4 agent or a DNA methyltransferase inhibitor, where the untreated subjects have, or are subject to developing, the same or similar disease, condition, symptom or the like.
  • Such increases or decreases may be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000% or more or within any range between any two of these values.
  • Modulation may be determined subjectively or objectively, e.g., by the subject's self-assessment, by a clinician's assessment or by conducting an appropriate assay or measurement, including, e.g., quality of life assessments, suitable assays for the level or activity of molecules, cells or cell migration within a subject and/or by modalities such as, but not limited to photographs, video, digital imaging and pulmonary function tests.
  • Modulation may be transient, prolonged or permanent or it may be variable at relevant times during or after an IL-4 agent or a DNA methyltransferase inhibitor is administered to a subject or is used in an assay or other method described herein or a cited reference, e.g., within times described infra, or about 12 hours to 24 or 48 hours after the administration or use of an IL-4 agent or a DNA methyltransferase inhibitor to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9 months or more after a subject(s) has received such treatment.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • polypeptide and the terms “protein” and “peptide” which are used interchangeably herein, refers to a polymer of amino acids.
  • exemplary polypeptides include gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.
  • sequence identity means that sequences are identical (i.e., on a nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino acid basis for polypeptides) over a window of comparison.
  • sequence identity is calculated by comparing two optimally aligned sequences over the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Methods to calculate sequence identity are known to those of skill in the art and described in further detail below.
  • stringent conditions or “stringent hybridization conditions” refer to conditions which promote specific hybridization between two complementary polynucleotide strands so as to form a duplex. Stringent conditions may be selected to be about 5 °C lower than the thermal melting point (Tm) for a given
  • polynucleotide duplex at a defined ionic strength and pH.
  • the length of the complementary polynucleotide strands and their GC content will determine the Tm of the duplex, and thus the hybridization conditions necessary for obtaining a desired specificity of hybridization.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the a polynucleotide sequence hybridizes to a perfectly matched complementary strand.
  • stringent hybridization conditions include a wash step of 0.2X SSC at 65 °C.
  • subject includes organisms which are capable of suffering from a lung injury, disease and/or disorder treatable by an IL-4 agent or a DNA methy transferase inhibitor (via direct administration of the IL-4 agent or DNA methyltransferase inhibitor to the subject or via treatment of cells with the IL-4 agent or DNA methyltransferase inhibitor, with such cells administered to the subject) or who could otherwise benefit from the administration of an IL-4 agent or a DNA methyltransferase inhibitor as described herein, such as human and non-human animals.
  • Preferred human animals include human subjects.
  • non-human animals includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • carrier refers to a diluent, excipient, and/or vehicle with which an active compound is administered.
  • the pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers are well known in the art.
  • the pharmaceutical compositions may also comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s) and so on.
  • the compositions can also contain other active components, e.g. other drugs for the treatment of lung injury or other diseases and/or conditions that are co-treated.
  • a cell is considered “allogeneic” with respect to another cell if both cells are derived from the same animal species but presents sequence variation in at least one genetic locus.
  • a cell is considered “allogeneic” with respect to a subject if the cell is derived from the same animal species as the subject but presents sequence variation in at least one genetic locus when compared to the subject's respective genetic locus.
  • a cell is considered “autologous” with respect to another cell if both cells are derived from the same individual or from genetically identical twins. A cell is considered
  • “autologous” to a subject if the cell is derived from the subject or a genetically identical twin. Autologous cells do not induce an immune reaction (such as a rejection) when they are introduced into an immuno-competent host.
  • a "suitable dosage level” refers to a dosage level that provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects. Often this dosage level is related to the peak or average serum levels resulting from administration of a drug at the particular dosage level.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the present invention provides isolated nucleic acid and/or polypeptide molecules having a nucleotide or polypeptide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98% or 99% identical to a polynucleotide or polypeptide comprising, consisting of, or consisting essentially of the polynucleotide or amino acid sequence of an IL-4 polynucleotide or polypeptide sequence as set forth herein, or fragments thereof.
  • nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the amino- or carboxy- terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences.
  • Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software are known in the art that may be used to obtain alignments of amino acid or nucleotide sequences.
  • One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al, Proc. Natl. Acad.
  • Gapped BLAST may be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).
  • BLAST-2 Altschul et al, Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences.
  • the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which incorporates the algorithm of Needleman and Wunsch (/.
  • Mol. Biol. (48):444-453 (1970)) may be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
  • the percent identity may be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art.
  • the default parameters of the alignment software are used.
  • the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.
  • whether any particular polynucleotide has a certain percentage sequence identity can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • Identity can exist over a region of the sequences that is at least about 5, at least about 10, about 20, about 40-60 residues in length or any integral value therebetween, or over a longer region than 60-80 residues, at least about 90-100 residues, or the sequences are substantially identical over the full length of the sequences being compared.
  • a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine,
  • phenylalanine, methionine, tryptophan methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s).
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Set USA 94:.412-417 (1997)).
  • polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • polypeptides and analogs can be further modified to contain additional chemical moieties not normally part of the protein.
  • Those derivatized moieties can improve the solubility, the biological half-life or absorption of the protein.
  • the moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).
  • the isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest (IL-4).
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81 :5662-5066 (1984) and U.S. Pat. No. 4,588,585.
  • ARDS acute respiratory distress syndrome
  • DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage.
  • DAC DNA methyltransferase inhibitor 5-aza-2'- deoxycytidine
  • Mice that received DAC exhibited accelerated resolution of their injury.
  • Lung CD4+CD25 hl Foxp3+ Tregs from DAC-treated wild type mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity.
  • mice did not resolve their injury in response to DAC.
  • Adoptive transfer of 2 x 10 5 DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. Additionally, in wild type mice having influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number.
  • DNA methyltransferase inhibition at least in part, augmented Treg number and functioned to accelerate repair of experimental lung injury. Epigenetic pathways were therefore identified as novel manipulable targets for the treatment of ARDS.
  • ARDS Acute respiratory distress syndrome
  • ARDS pathology Despite extensive investigation into initial events that cause ARDS pathology, no targeted therapies have been previously identified to promote repair in the damaged lung. Resolution of inflammation following acute lung injury has been identified to be an active process (2) that required CD4+CD25+Foxp3+ regulatory T cells (Tregs) in a direct lung injury mouse model:
  • LPS lipopolysaccharide
  • surviving wild type mice spontaneously resolved their injury 7-10 days after receiving LPS.
  • Lymphocytes did not determine initial injury severity; however, injury resolution required lymphocytes, as evidenced by unremitting LPS -induced lung inflammation in lymphocyte-deficient recombinase activating gene- 1-null (Rag-l 7 ) mice.
  • the Treg subpopulation was identified as the active lymphocyte fraction involved in promoting resolution via pro-repair effects on macrophage inflammatory function, neutrophil efferocytosis, epithelial proliferation (4), and limitation of fibroproliferation (5).
  • Tregs have also been characterized as promoting repair in other tissue damage models, as well as in clinical scenarios (6,7). While Treg cell transfer may be a viable management strategy for chronic conditions (7), the briskly evolving nature of ARDS makes a cell transfer-based therapy impractical.
  • Tregs comprise a CD4+ lymphocyte subset that attenuates both innate and adaptive immune responses (9).
  • Treg development and function require epigenetic programming— prominently through DNA demethylation— and forkhead box protein 3 (Foxp3) transcription factor expression (10, 11).
  • Constitutive Foxp3 expression was identified as necessary for Treg suppressive activity (12, 13), and epigenetic marks were characterized as regulating transcription at the Foxp3 locus and the loci of other key Treg genes (10,14).
  • Treg gene loci including Foxp3
  • CpG cytosine-phospho-guanine
  • Foxp3 expression correlated with Treg suppressive function (11,20,21).
  • DNMTs DNA methyltransferases
  • CpG methylation within promoters and transcription factor binding sites represses gene transcription, and DNMT inhibition or knockdown can lead to DNA demethylation.
  • DNMTs determine Foxp3 expression and Treg identity, as DNMT silencing or inhibition via siRNA or 5 -aza-2' -deoxycytidine (DAC) have respectively been identified to lead to Foxp3 expression and Treg phenotype in naive CD4+ non-Treg cells (17, 23-25).
  • DAC 5 -aza-2' -deoxycytidine
  • Tregs Foxp3+ regulatory T cell DNA methyltransferase inhibition was established as an epigenetic mechanism that accelerates resolution of acute lung injury.
  • ARDS is a devastating disease for which there is a lack of targeted therapy despite extensive insight into the initial inflammatory injury.
  • Events determining resolution of lung inflammation were a main focus of the studies performed herein. Resolution of injury required not only cessation of ongoing pathology but also active repair of damaged tissues (2).
  • resolution was identified to be accelerated by a DNMT inhibitor administered after lung injury establishment. DNMT inhibition did not modify early LPS- injury as described herein, highlighting that dynamic changes in DNA methylation patterns that likely occur during resolution.
  • the sterile inflammatory model described herein has relevance for many ARDS causes including aspiration of gastric contents, ventilator-induced lung injury, near drowning, and collateral lung injury associated with treated bacterial infection. Moreover, the data described herein, obtained using an influenza model, broadens the applicability of epigenetic manipulation as a therapeutic strategy for ARDS. Diphtheria toxin-treated WT mice were selected as controls for the experiments described herein using Foxp3DTR mice, which expressed a normal Foxp3 protein and a diphtheria toxin receptor-green fluorescent protein fusion product (DTR-GFP).
  • DTR-GFP diphtheria toxin receptor-green fluorescent protein fusion product
  • mice expressing a Foxp3-GFP fusion protein have facilitated studies of Treg biology (44), these mice exhibited abnormal Treg epigenetic programming due to the altered Foxp3 protein (45).
  • mice with a normal Foxp3 protein were selected to ensure fidelity of epigenetic responses.
  • Tregs coordinate resolution of direct lung injury via cellular interactions that lead to pro-repair effects on alveolar macrophage responses (3), epithelial regeneration (4), and limitation of fibrocyte-mediated fibrosis (5).
  • the specific Treg subset involved in injury resolution has remained undefined.
  • Ex vivo treatment of Tregs with DAC followed by adoptive transfer to LPS-injured Treg-depleted animals increased BAL fluid active TGF- ⁇ concentration, as compared to ex vivo vehicle treatment.
  • Tregs required TGF- ⁇ to effect repair following LPS injury (3), which indicated that peripherally induced Tregs (pTregs or iTregs) were likely the responsible fraction.
  • thymus -derived natural Treg (tTreg or nTreg) expansion or recruitment might also have contributed to the lymphocyte response after lung injury, as adoptive transfer of 1 x 10 6 splenic nTregs mediated resolution in lymphocyte deficient mice (3,5).
  • the CpG methylation signature of Treg gene loci distinguished committed thymus-derived Tregs and TGF- -induced Tregs (10). The results were somewhat limited in that region-specific CpG methylation patterns were not measured directly; however, others have previously described that Treg induction in the presence of a DNMT inhibitor generated a thymus-derived Treg epigenetic profile (10,8,25). Lung Treg CpG methylation pattern analysis paired with gene expression profiling is currently pursued to identify the significance of Treg epigenetic signatures following injury and with DNMT inhibitor treatment.
  • Treg CD39 surface expression increased in response to DAC after lung injury, indicating that extracellular ATP hydrolysis was involved as a mechanism by which Tregs exerted their pro-repair program following DNMT inhibition. Damaged cells released ATP into the extracellular milieu, where it exhibited multiple pro-inflammatory effects; CD39+ Treg-mediated ATP hydrolysis was identified to restore homeostasis to injured tissues (31). DAC did not increase Treg CD39 expression in the absence of inflammation— in vitro ( Figure 6) or following i.t. water. However, lung Treg CD39 expression increased following adoptive transfer of DAC-treated Tregs to an injured host.
  • DNMT inhibition increased lung Treg frequency and suppressive phenotype and functioned to promote resolution.
  • a therapeutic approach to treatment of lung injury has therefore been identified, and additional investigation into the epigenetic marks and mechanisms underlying the findings described herein are expected to further enhance therapeutic options for patients with ARDS and other acute inflammatory conditions.
  • the instant invention involves contacting Treg cells with a DNA methyltransferase inhibitor, to promote therapeutic effects of such treated Treg cells.
  • Treg subpopulation as the active lymphocyte fraction involved in promoting repair, via its effects on macrophage function, neutrophil efferocytosis, epithelial proliferation (4), and limitation of fibroproliferation (5).
  • Tregs also had also been identified as promoting repair in other tissue damage models as well as clinical scenarios (6, 7). While Treg adoptive transfer may be a viable
  • Tregs comprise a CD4+ lymphocyte subset that suppresses both innate and adaptive immune responses (9). Treg development and function require both epigenetic
  • Tregs coordinate repair following direct lung injury via effects on alveolar macrophage pro-inflammatory responses, neutrophil efferocytosis (3), alveolar epithelial regeneration (4), and limitation of fibrosis (5).
  • a key Treg feature— DNA demethylation— has been newly exploited to increase Treg frequency and suppressive phenotype and function following lung injury. While the specific Treg subset involved in injury resolution remains undefined, remarkable amelioration of recovery from acute lung injury was observed in performing the methods of the invention.
  • Tregs require transforming growth factor beta (TGF- ⁇ ) to effect repair following LPS injury (3), which has suggested that peripherally induced Tregs (pTregs or iTregs) may be the responsible fraction.
  • TGF- ⁇ peripherally induced Tregs
  • pTregs or iTregs peripherally induced Tregs
  • thymus-derived natural Treg (tTreg or nTreg) expansion and/or recruitment might also have contributed to the lymphocyte response after lung injury, as adoptive transfer of splenic nTregs mediated resolution in lymphocyte deficient mice (3,5).
  • the CpG methylation signature of Treg gene loci distinguishes committed thymus-derived Tregs and TGF- -induced Tregs (10).
  • Treg induction in the presence of a DNMT inhibitor also generated a thymus-derived Treg epigenetic profile (8,10,25).
  • Treg epigenetic signatures following injury can be further surveyed by identifying lung Treg CpG methylation patterns, paired with gene expression profiling.
  • Treg CD39 surface expression increased in response to DAC after lung injury, indicating extracellular ATP hydrolysis as a likely mechanism by which Tregs exerted their pro-repair program following DNMT inhibition. Damaged cells released ATP into the extracellular milieu, where it exhibited multiple pro-inflammatory effects; CD39+ Treg-mediated ATP hydrolysis can restore homeostasis to injured tissues (31). DAC did not increase Treg CD39 expression in the absence of inflammation— in vitro ( Figure 6) or following i.t. water (data not shown). However, lung Treg CD39 expression increased following adoptive transfer of DAC-treated Tregs to an injured host.
  • CTLA-4 was modestly up-regulated with DAC treatment; these nominal increases might be significant, as CTLA-4 transmits a potent negative signal in the immunological synapse (32).
  • CD25 expression was not modified by DNMT inhibition, potentially due to minimal pre-existing methylation at the Treg I12ra gene locus (10).
  • Treg Ki-67 expression increased in response to DAC, indicating an increased proliferative capacity.
  • lung Treg frequency increased over 2- fold in DAC-treated mice compared with vehicle five days post-LPS, and DAC treatment led to a robust proliferation following adoptive transfer.
  • DNMTs DNA methyltransferases
  • the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine accelerated resolution of experimental lung injury via a salutary effect on Treg phenotype and function. Epigenetic manipulation of the regulatory T cell lineage was thus identified as an attractive therapeutic strategy for the acute respiratory distress syndrome and other acute inflammatory disorders.
  • Acute respiratory distress syndrome is a common and lethal inflammatory condition without effective pharmacotherapy.
  • CD4+CD25+Foxp3+ regulatory T cells resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage have heretofore remained unknown.
  • DNA methyltransferases repress gene transcription by catalyzing DNA methylation at CpG residues within gene promoters and transcription factor binding sites.
  • DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage.
  • DNMT inhibition increases lung Treg frequency and suppressive phenotype and function after injury; moreover, Tregs mediated the pro-resolution effect of DNMT inhibition.
  • Epigenetic pathways represent novel manipulable targets for the treatment of the acute respiratory distress syndrome. The epigenetic marks and mechanisms underlying the current findings promote contemplation of therapeutic options for patients with the acute respiratory distress syndrome and other acute inflammatory conditions.
  • ARDS acute respiratory distress syndrome
  • intratracheal lipopolysaccharide or Pseudomonas bacteria administration was used to model experimental acute lung injury (ALI) and to further understand mediators of the resolution phase of ARDS.
  • ALI acute lung injury
  • Recent work has demonstrated that macrophages transition from a predominant pro-inflammatory Ml phenotype during acute inflammation to an anti-inflammatory M2 phenotype with ALI resolution.
  • IL-4 a potent inducer of M2-specific protein expression, would accelerate ALI resolution and lung repair through reprogramming of endogenous inflammatory macrophages was tested.
  • IL-4 treatment was found to offer dramatic and surprising benefits following delayed administration to mice that had been subjected to experimental ALI, including increased survival, accelerated resolution of lung injury, and improved lung function.
  • expression of the M2 proteins Argl, FIZZl and Yml was increased in lung tissues following IL-4 treatment, and among macrophages, FIZZl was most prominently upregulated in the interstitial subpopulation.
  • MMR macrophage mannose receptor
  • Dectin- 1 was a similar trend was observed for the expression of macrophage mannose receptor (MMR) and Dectin- 1 on the surface of alveolar macrophages following IL-4 administration. Macrophage depletion or STAT6 deficiency abrogated the therapeutic effect of IL-4.
  • ARDS Acute respiratory distress syndrome
  • Lung inflammation has been described as intimately associated with a phenotypically and functionally diverse set of monocytes and macrophages (69, 81, 95).
  • Early macrophage classification of phenotypes included Ml (classically-activated or pro-inflammatory) or M2 (alternatively- activated or anti-inflammatory).
  • Ml classically-activated or pro-inflammatory
  • M2 alternatively- activated or anti-inflammatory
  • macrophages become Ml, possess strong microbiocidal activity, and secrete high levels of pro-inflammatory cytokines.
  • persistence of Ml macrophages can be detrimental to wound healing (96).
  • M2 macrophage transformation from a predominant Ml phenotype during acute inflammation to a pro-resolution, M2 phenotype with initiation of lung repair and restoration of tissue homeostasis (56, 73).
  • M2 macrophages have been identified as elicited by IL-4 and/or IL-13 in a STAT6-dependent manner, and have been commonly identified in the mouse by surface expression of mannose receptor, and intracellular expression of arginase-1 (Argl), chitinase-like 3 (Yml), and FIZZl (Relma) (68).
  • M2 macrophages have been believed to be important in wound healing and can promote tissue repair by limiting Th2- associated cellular inflammation, cytokine production, and fibroproliferation (86, 90, 91). Yet, a causal role for pro-resolution M2 macrophages during ALI resolution and lung repair has not previously been established.
  • IL-4 therapy decisively and surprisingly accelerated resolution of sterile and infection-induced lung inflammation, and required macrophages and Stat6 expression to orchestrate this response.
  • IL-4 improved mortality, accelerated resolution of lung injury, and restored lung function.
  • Lung macrophages and STAT6 transcription factor expression were necessary for IL-4 to exert its therapeutic effects on lung injury resolution; Tregs, on the other hand, did not appear to be required.
  • the M2 proteins Argl, FIZZl, and Yml were increased in the lung with IL-4 treatment, and among macrophages FIZZl upregulation was most pronounced in the interstitial sub-population.
  • the importance of the interstitial macrophage sub-population towards IL-4-derived ALI resolution was further supported by their preferential depletion in intravenous liposomal clodronate experiments.
  • Mitigating influx and enhancing removal of accumulated alveolar neutrophils has been identified as a hallmark of resolution of lung inflammation (79).
  • M2 macrophages have been identified as effective at phagocytosis (61, 73), and IL-4 has been identified to restore impaired macrophage phagocytosis (64).
  • IL-4 therapy resulted in a several-fold reduction in alveolar neutrophils at later time points. Therefore, a likely IL-4-mediated benefit is to enhance macrophage efferocytosis of apoptotic neutrophils to accelerate experimental ALI resolution.
  • IL-4 therapy reduced whole lung collagen, indicating that it likely also regulated fibroproliferation.
  • Ml macrophages have been identified as typically associated with enhanced microbial phagocytosis and killing of intracellular bacteria via iNOS, TNF-a, IL-12 production (73), IL-4 reprogramming of M2 macrophages was observed to accelerate ALI resolution in the infection model described herein, without any apparent detrimental effects on bacterial clearance. M2 macrophages were reported to possess increased phagocytic activity via upregulation of Fc or scavenger receptors (61).
  • macrophages have been characterized as a highly plastic cell, which therefore would likely reprogram to a predominant Ml phenotype in response to infection-induced signaling of pattern recognition receptors.
  • a significant percentage of quiescent alveolar macrophages have been identified to display an M2 phenotype with prominent mannose receptor expression (85, 89, 105), yet have exhibited undiminished capacity to initiate a robust pro-inflammatory response to infectious and noninfectious stimuli. Therefore, in some embodiments, not only has IL-4 therapy been identified to accelerate ALI resolution and lung repair, it likely has also promoted homeostasis by reprogramming lung macrophages back to their quiescent, predominantly M2 phenotypic state.
  • Tregs can resolve lung inflammation by abrogating pro-inflammatory macrophage responses and, like IL-4, by reprogramming macrophages towards an M2 phenotype (59). Indeed, the work by Taams and colleagues showed that human Tregs dampened LPS-induced Ml monocyte pro-inflammatory responses while promoting an M2 phenotype (99, 101). Moreover, the prospect that Tregs provide an important IL-4 source, and as such, likely regulate macrophage phenotype and function during lung inflammation as a mechanism to support endogenous resolution, should not be excluded.
  • IL-4 treatment is expected to obviate the need for endogenous Treg-derived IL-4, likely explaining the lack of Treg requirement. Noting the ability of IL-4 to induce endogenous Treg proliferation and maintain their suppressive function (87), in some embodiments, IL-4 treatment is expected to exert a synergistic pro-repair effect by programming macrophages and Tregs.
  • IL-4 treatment was used instead of IL-13, the latter of which has also been described to signal through a macrophage IL-4 receptor to promote M2 protein expression.
  • IL-4 has previously been described to induce more robust expression of M2 genes (72) and is also believed to be less likely to induce fibrosis (73).
  • IL-4- or IL-13-derived M2 macrophages there are several other subtypes of anti-inflammatory macrophages that are not addressed here yet would be predicted as important for endogenous repair pathways (56).
  • Recent work has identified macrophages derived from stimulation with high-density immune complexes as expressing elevated levels of IL-10 and protecting from lethal endo toxemia in a STAT6-independent fashion (66).
  • IL-6/IL-10/STAT3- and TLR/MyD88/C/EBP Signaling pathways that do not require STAT6 to induce M2 marker expression
  • IL-6/IL-10/STAT3- and TLR/MyD88/C/EBP both have also been described as of significance in experimental ALI (60, 92).
  • the therapeutic benefits of IL-4 and M2 marker expression are expected to be amplified by similar STAT6-independent autocrine or paracrine signals.
  • IL-4 signaling through macrophage STAT6 appeared necessary to accelerate ALI resolution, indicating that the specific cytokine activity was critical.
  • IL-4 a novel therapeutic agent that has been newly described to resolve sterile and non-sterile lung inflammation robustly, has been identified.
  • IL-4 treatment was administered as an effective rescue therapy, well after the onset of experimental ALI, indicating translational potential for other injuries, diseases and/or disorders.
  • the present invention is based, at least in part, upon identification of modulation of macrophages - specifically, enhancing transition of lung (e.g. alveolar) macrophages from an Ml phenotype to M2 phenotype - as a factor underlying accelerated recovery from acute lung injury of IL-4-treated subjects.
  • exemplary references that have identified macrophages as critical for resolution of lung inflammation include:
  • D'Alessio FR Tsushima K, Aggarwal NR, Mock JR, Eto Y, Garibaldi BT, Files DC, Avalos CR, Rodriguez JV, Waickman AT, Reddy SP, Pearse DB, Sidhaye VK, Hassoun PM, Crow MT, King LS. Resolution of experimental lung injury by monocyte-derived inducible nitric oxide synthase. Immunol. 2012 Sep 1 ; 189(5):2234-45.
  • IL-4 specifically recombinant forms of IL-4, was identified to accelerate recovery from acute lung injury.
  • exemplary forms of recombinant IL-4 include recombinant murine IL-4, which is a 13.5 kDa globular protein containing the following 121 amino acid residues, derived from an E. coli source:
  • An example of a corresponding human IL-4 sequence is that of recombinant human IL-4, chain A, locus 1ITL, having accession number GI:157831503, which is a 15.1 kDa globular protein containing amino acid residues manufactured using all non-animal reagents: MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSH HEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLE RLKTIMREKYSKCSS (SEQ ID NO: 3).
  • the nucleotide sequence of human recombinant IL-4 is: 5'- ATGCACATACACGGATGCGACAAAAATCATCTTCGGGAGATTATTGGGATCCTG AACGAGGTGACGGGAGAAGGAACCCCATGCACCGAGATGGACGTCCCCAACGTT CTGACTGCCACTAAGAACACAACTGAAAGTGAACTCGTATGCCGCGCATCGAAG GTTTTGCGAATATTTTACCTTAAGCACGGGAAGACTCCGTGCTTGAAAAAGAACT CATCGGTTCTCATGGAATTGCAAAGACTTTTCCGCGCCTTCCGGTGCCTCGACTC GTCTATTTCTTGCACTATGAACGAGTCAAAAAGTACCAGTCTTAAAGATTTCCTC GAAAGCCTTAAGAGTATAATGCAGATGGATTATAGC-3' (SEQ ID NO: 4).
  • Variant forms of human IL-4 polypeptide are also known in the art, including those of GenBank Accession Nos. 1HIJ_A GI: 157831337 (chain A, Interleukin-4 mutant with Arg 88 replaced with Gin (R88q)); 2D48_A GI: 109157435 (chain A, crystal structure of the Interleukin-4 variant T13d); 1HZI_A GI: 15826610 (chain A, Interleukin-4 mutant E9a); and 2B8Z_A GI: 109157203 (chain A, crystal structure of the Interleukin-4 variant R85a).
  • Mouse IL-4 niRNA consists of the sequence of GenBank Accession No. M25892.1 GL533236, while an exemplary human IL-4 mRNA sequence is that of GenBank Accession No. M13982.1 GL186334.
  • IL-4 in recombinant form is safe for human use and can be used as immunotherapy: Ghoreschi K, Thomas P, Breit S, Dugas M, Mailhammer R, Van Eden W, et al. Interleukin-4 therapy of psoriasis induced Th2 responses and improves human autoimmune disease. Nat Med 2003;9:40-46.
  • IL-4Ra agonists of IL-4Ra are also contemplated for inclusion in the pharmaceutical compositions and use in the methods of the instant invention.
  • exemplary forms of IL-4 Ra agonists include mutein forms of IL-4 as set forth in Shanafelt et al. Proc. Natl. Acad. Sci. USA 95: 9454-94, while activating antibodies directed to IL-4Ra are also contemplated.
  • Interleukin 4 is a cytokine that induces differentiation of naive helper T cells (ThO cells) to Th2 cells. Upon activation by IL-4, Th2 cells subsequently produce additional IL-4 in a positive feedback loop. The cell that initially produces IL-4, thus inducing ThO differentiation, has not been identified, but recent studies suggest that basophils may be the effector cell. It is closely related and has functions similar to Interleukin 13. It has many biological roles, including the stimulation of activated B-cell and T-cell proliferation, and the differentiation of B cells into plasma cells. It is a key regulator in humoral and adaptive immunity. IL-4 induces B-cell class switching to IgE, and up-regulates MHC class II production. IL-4 decreases the production of Thl cells, macrophages, IFN-gamma, and dendritic cell IL-12. Overproduction of IL-4 is associated with allergies.
  • Tissue macrophages play an important role in chronic inflammation and wound repair.
  • the presence of IL-4 in extravascular tissues promotes alternative activation of macrophages into M2 cells and inhibits classical activation of macrophages into Ml cells.
  • An increase in repair macrophages (M2) is coupled with secretion of IL-10 and TGF- ⁇ that result in a diminution of pathological inflammation. Release of arginase, proline, polyaminases and TGF- ⁇ by the activated M2 cell is tied with wound repair and fibrosis.
  • the receptor for Interleukin-4 is known as the IL-4Ra. This receptor exists in 2 different complexes throughout the body. Type 1 receptors are composed of the IL-4Ra subunit with a common ⁇ chain and specifically bind IL-4. Type 2 receptors consist of an IL- 4Ra subunit bound to a different subunit known as IL-13Ral, and have the ability to bind both IL-4 and IL-13, two cytokines with closely related biological functions. IL-4 also has been shown to drive mitogenesis, dedifferentiation, and metastasis in rhabdomyosarcoma. IL-4, along with other Th2 cytokines, is involved in the airway inflammation observed in the lungs of patients with allergic asthma.
  • Acute lung injury caused by a wide variety of insults results in increased pulmonary capillary permeability and pulmonary edema without any increase in capillary or left atrial pressures: so called "low pressure edema" or ARDS.
  • This is the single most common pulmonary complication of ICU patients, and accounts for a tremendous burden of morbidity and mortality.
  • Acute respiratory distress syndrome (ARDS), the clinical correlate of severe acute lung injury (ALI) in humans, is an important cause of morbidity and mortality in critically ill patients.
  • Acute respiratory distress syndrome (ARDS) is a devastating disease hallmarked by acute inflammation of the lungs, and results in severe lung damage and an attributable mortality of 30-40%. Yet there is no effective therapy. Infectious etiologies, such as sepsis and pneumonia (including influenza and SARS), are leading causes of ALI/ ARDS.
  • ALI/ ARDS in humans is characterized by a severe acute inflammatory response in the lungs and neutrophilic alveolitis.
  • Inflammatory stimuli from microbial pathogens such as endotoxin (lipopolysaccharide, LPS)
  • LPS lipopolysaccharide
  • ARDS The physiological hallmark of ARDS is disruption of the alveolar-capillary membrane barrier (i.e., pulmonary vascular leak), leading to development of non-cardiogenic pulmonary edema in which a proteinaceous exudate floods the alveolar spaces, impairs gas exchange, and precipitates respiratory failure. Both alveolar epithelial and endothelial cell injury and/or death have been implicated in the pathogenesis of ALI/ ARDS (1).
  • ICU intensive care unit
  • ARDS is a complex clinical syndrome which is initiated by injury to the lung, often in the setting of pneumonia and/or sepsis, and aggravated by ventilator-induced injury.
  • LPS bacterial endotoxin
  • TLR4 Toll-like receptor 4
  • Contemplated treatments for ARDS include treatments that involve decreasing lung inflammation, decreasing septal edema, decreasing alveolar and/or endothelial inflammation, or alleviating another symptom of the ARDS.
  • Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) were defined by a panel of experts in 2011 (an initiative of the European Society of Intensive Care Medicine endorsed by the American Thoracic Society and the Society of Critical Care Medicine) as the Berlin Definition. Presently there are three stages: mild, moderate, and severe with an associated increased mortality (27%; 95% CI, 24%-30%; 32%; 95% CI, 29%- 34%; and 45%; 95% CI, 42%-48%, respectively; P ⁇ .001) and increased median duration of mechanical ventilation in survivors (5 days; interquartile [IQR], 2-11; 7 days; IQR, 4-14; and 9 days; IQR, 5-17, respectively; P ⁇ .001).
  • the definition was empirically evaluated using patient- level meta-analysis of 4188 patients with ARDS from 4 multicenter clinical data sets and 269 patients with ARDS from 3 single-center data sets containing physiologic information.
  • the categories of ARDS are based on the degree of hypoxemia determined by the ratio of Pa02/Fi02 where the Pa02 is the partial pressure of oxygen in arterial blood and the Fi02 is the fraction of inspired oxygen.
  • the categorization is as follows: (1) Mild ARDS: 200 mm Hg ⁇ Pa02/Fi02 and less than or equal to 300 mm Hg; (2) Moderate ARDS: 100 mm Hg ⁇ Pa02/Fi02 and less than or equal to ⁇ 200 mm Hg; and (3) Severe ARDS: Pa02/Fi02 is less than or equal to 100 mm Hg.
  • ARDS The causes of ARDS have been enumerated, with the most common being: sepsis, aspiration, pneumonia, severe trauma (bilateral lung contusion, fat embolism after long bone fracture, sepsis that develops several days after severe trauma or burns, and massive traumatic tissue injury), massive transfusion, transfusion related acute lung injury, lung and hematopoietic stem cell transplantation, drugs and alcohol, and genetic determinants such as mutations in the surfactant protein B (SP-B) gene.
  • SP-B surfactant protein B
  • Management of ARDS includes treatment of the underlying condition, mechanical or noninvasive ventilation, fluid and hemodynamic therapy, treatment of opportunistic infection, nutrition, and pharmacologic therapy.
  • glucocorticoids glucocorticoids, alprostadil, surfactant, ketoconazole, N-acetylcysteine, procysteine, lisofylline, and site-inactivated recombinant factor Vila.
  • ARDS persists as a devastating disease with no effective pharmacotherapies despite extensive insight into the initial inflammatory injury (36). Therefore, the examples described herein focused on events determining resolution of lung inflammation. Resolution of injury requires not only cessation of ongoing pathology but also active repair of damaged tissues (2,37). Here, it was shown that resolution could be accelerated by an IL-4 agent or a small molecule administered after lung injury establishment. DNMT inhibition did not modify early injury in the studies described herein, highlighting that dynamic changes in DNA methylation patterns likely occurred during resolution.
  • Lipopolysaccharide-induced pulmonary inflammation is a well-known and well documented animal model for ARDS. Measures of extent of inflammation include cell counts from bronchoalveolar lavage (BAL) and a measure of pro-inflammatory cytokine levels in BAL fluid and lung parenchymal homogenates. LPS-induced permeability in the lung (i.e. extent of Acute Lung Injury) can also be measured.
  • BAL bronchoalveolar lavage
  • LPS-induced permeability in the lung i.e. extent of Acute Lung Injury
  • An IL-4 agent of the invention can be administered to a subject at any time after a lung injury event and/or after diagnosis of a lung injury or other disease or disorder in a subject.
  • an IL-4 agent of the invention is administered to a subject at least 2-3 hours after the lung injury event, at least 6 hours after the lung injury event, at least 12 hours after the lung injury event, or at least 24 hours after the lung injury event.
  • an IL-4 agent of the invention is administered to a subject at least 36 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days after the lung injury event.
  • an IL-4 agent of the invention is administered to a subject within 1-3 or 2-3 hours, 6, 12, 18 or 24 hours after diagnosis of a lung injury or of an acute inflammatory disease or disorder.
  • an IL-4 agent of the invention is administered within 36 hours of diagnosis, within 48 hours of diagnosis, within 72 hours of diagnosis, within four days of diagnosis, within five days of diagnosis, within six days of diagnosis, within seven days of diagnosis, or at more than seven days from diagnosis of a lung injury or of an acute inflammatory disease or disorder.
  • Dramatic improvement/recovery from a lung injury, acute inflammatory disease or disorder or influenza are noted/are predicted to occur in instances where treatment with an IL-4 agent of the invention occurs, e.g., 24-48 or more hours after a lung injury event and/or onset of an acute inflammatory disease or disorder or influenza.
  • a DNA methyltransferase inhibitor of the invention can be administered to a subject at any time after a lung injury event and/or after diagnosis of a lung injury or other disease or disorder in a subject.
  • a DNMT inhibitor of the invention is administered to a subject at least 2-3 hours after the lung injury event, at least 6 hours after the lung injury event, at least 12 hours after the lung injury event, or at least 24 hours after the lung injury event.
  • a DNMT inhibitor of the invention is administered to a subject at least 36 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days after the lung injury event.
  • a DNMT inhibitor of the invention is administered to a subject within 1-3 or 2-3 hours, 6, 12, 18 or 24 hours after diagnosis of a lung injury or of an acute inflammatory disease or disorder.
  • a DNMT inhibitor of the invention is administered within 36 hours of diagnosis, within 48 hours of diagnosis, within 72 hours of diagnosis, within four days of diagnosis, within five days of diagnosis, within six days of diagnosis, within seven days of diagnosis, or at more than seven days from diagnosis of a lung injury or of an acute inflammatory disease or disorder.
  • a cultured cellular preparation can be administered to the subject in a therapeutically effective amount.
  • a T cell population containing Treg cells is obtained (e.g., via isolation from a subject) and is optionally cultured in vitro, with the cell population contacted with a DNA methyltransferase inhibitor to promote Treg activity and/or an expanded Treg cell population.
  • treated cells are (re)introduced into the pulmonary circulation by infusion of the cells either into a peripheral vein or a central vein, from where they move with the circulation to the pulmonary system, and become lodged in the smallest arterioles of the vascular bed of the lungs.
  • Direct injection into the pulmonary circulation can also be adopted, for example through a Swan Ganz catheter.
  • Injection into the right ventricle or right atrium may be carried out using the pacing port of a Swan Ganz catheter.
  • the infusion can optionally be done in a bolus form i.e.
  • injection of all the cells during a short period of time or it may be accomplished by a continuous infusion of small numbers of cells over a long period of time, or alternatively by administration of limited size boluses on several occasions over a period of time.
  • introduction of treated cells into the lungs can also be accomplished through inhalation of the cells using known pulmonary administration methods, such as an inhaler.
  • the transferred, treated cells of ex vivo therapy applications of the invention can themselves largely or completely be retained in the pulmonary circulation, and especially in the arterioles of the patient's lungs, the expression products of the transgenes thereof are not restricted in this manner. They can be expressed and secreted from the treated cells, and travel through the normal circulation of the patient to other, downstream body organs where they can exert a therapeutic effect.
  • the treated cells can optionally express products designed for treatment of other body organs of the patient. Such products expressed in the pulmonary system will target the other, predetermined organs and be delivered thereto by the natural circulation system of the patient.
  • an alternative means of increasing the Treg/pro- inflammatory T cell ratio involves the administration of the supernatant of a treated cell culture of Treg cells to a subject.
  • the cell culture supernatant can comprise certain cells or fractions (for example a part of the cytoplasmic membrane).
  • testing for the possibility and/or development of an immune response is performed. It is possible to determine if two cells are considered immunogenic with respect to one another by conducting conventional in vitro assays, such as a mixed lymphocyte reaction. It is also expected that MHC-disparate cells would be considered immunogenic with respect to one another.
  • the cell culture supernatant apart from being optionally filtered to remove cells and cellular debris, is not submitted to further extraction/size fractionation prior to its administration to the subject.
  • the cell culture supernatant thus comprises the conditioned media from the cell culture (e.g. cellular byproducts including the cytokines secreted by the cultured cells).
  • An alternative way of increasing the Treg/pro-inflammatory Tcell ratio in a subject to be treated is to administer the plasma of an animal (such as a rodent) that has been treated with a DNMT inhibitor.
  • this plasma can comprise Treg cells of the animal or a derivative therefrom (a part of the cytoplamsic membrane from the Treg cell, for example).
  • Processes for obtaining the plasma from an animal are known to those skilled in the art and usually include a cell lysis (to remove erythrocytes) as well as centrifugation.
  • the plasma apart from being optionally filtered to remove cells and cellular debris, is not submitted to further extraction/size fractionation prior to its administration to the subject.
  • the plasma thus comprises the cellular by-products generated upon the treatment of Treg cells with a DNMT inhibitor (including the cytokines produced by the immune system).
  • the transferred, treated cells of ex vivo therapy applications of the invention can themselves largely or completely be retained in the pulmonary circulation, and especially in the arterioles of the patient's lungs, the expression products of the transgenes thereof are not restricted in this manner. They can be expressed and secreted from the treated cells, and travel through the normal circulation of the patient to other, downstream body organs where they can exert a therapeutic effect.
  • the treated cells can optionally express products designed for treatment of other body organs of the patient. Such products expressed in the pulmonary system will target the other, predetermined organs and be delivered thereto by the natural circulation system of the patient.
  • Mammalian cells such as the patient's own (i.e. autologous) or cells from another individual (i.e. allogeneic) cells can be cultured in vitro and treated with a DNMT inhibitor. Then the treated cells are introduced into the patient, so that the treated Treg cells can exert their effect in the body, for therapeutic purposes.
  • DNMT inhibitor treatment of a T cell-containing population selective culturing of (active) Treg cells can be performed, so that administration of the cells to the patient can be limited to the (active) Treg. In other cases, all of the cells subject to the DNMT inhibitor treatment are administered.
  • the present invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising an IL-4 agent or a DNA methyltransferase inhibitor employed in the present invention.
  • the IL-4 agent or DNA methyltransferase inhibitor targeting lung injury can be suitably formulated and introduced into a subject or the environment of a cell by any means recognized for such delivery.
  • compositions typically include the agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • compositions of the invention could also be formulated as nanoparticle formulations.
  • the compounds of the invention can be administered for immediate -release, delayed- release, modified-release, sustained-release, pulsed-release and/or controlled-release applications.
  • compositions of the invention may contain from 0.01 to 99% weight - per volume of the active material.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of an IL-4 agent or a DNA methyltransferase inhibitor targeting lung injury depends on the IL- 4 agent or DNA methyltransferase inhibitor selected.
  • an effective dosage depends on the IL- 4 agent or DNA methyltransferase inhibitor selected.
  • single dose amounts of an IL-4 agent or a DNA methyltransferase inhibitor targeting lung injury in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 ⁇ g, or 10, 30, 100, or 1000 mg may be administered.
  • 1-5 g of the compositions can be administered.
  • a therapeutically effective amount of the compound of the present invention can be determined by methods known in the art.
  • the therapeutically effective quantities of a pharmaceutical composition of the invention will depend on the age and on the general physiological condition of the patient and the route of administration.
  • the therapeutic doses will generally be between about 10 and 2000 mg/day and preferably between about 30 and 1500 mg/day. Other ranges may be used, including, for example, 50-500 mg/day, 50-300 mg/day, 100-200 mg/day.
  • Administration may be once a day, twice a day, or more often, and may be decreased during a maintenance phase of the disease or disorder, e.g. once every second or third day instead of every day or twice a day.
  • the dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art.
  • certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of an IL-4 agent or a DNA methyltransferase inhibitor targeting lung injury can include a single treatment or, optionally, can include a series of treatments.
  • Suitable amounts of an IL-4 agent or a DNA methyltransferase inhibitor targeting lung injury must be introduced and these amounts can be empirically determined using standard methods.
  • Exemplary effective concentrations of individual IL-4 agent or DNA methyltransferase inhibitor species in the environment of a cell can be 500 millimolar or less, 50 millimolar or less, 10 millimolar or less, 1 millimolar or less, 500 nanomolar or less, 50 nanomolar or less, 10 nanomolar or less, or even compositions in which concentrations of 1 nanomolar or less can be used.
  • compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a lung injury.
  • Treatment or “treating” as used herein, is defined as the application or
  • a therapeutic agent e.g., an IL-4 agent or a DNA methyltransferase inhibitor
  • administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the injury, (or, where an isolated tissue or cell line is used, from a subject not having the injury, e.g., for transfer to isolated tissue, cell or supernatant to a patient having the injury) for a symptom of the injury, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the injury and/or the symptoms of the injury.
  • a therapeutic agent e.g., an IL-4 agent or a DNA methyltransferase inhibitor
  • DNA methyltransferase inhibition markedly enhanced Treg function to accelerate lung injury repair in a mouse model.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed.
  • Garibaldi BT D'Alessio FR, Mock JR, Files DC, Chau E, Eto Y, Drummond MB, Aggarwal NR, Sidhaye V, King LS. Regulatory T cells reduce acute lung injury
  • Wilson CB Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol 2009;9:91-105.
  • Fadok VA Bratton DL
  • Konowal A Freed PW
  • Westcott JY and Henson PM.
  • CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human
  • DAC 5-aza-2'-deoxycytidine
  • mice C57BL/6 wild type (WT) and Rag- ⁇ 7" mice were purchased from Jackson Laboratory (Bar Harbor, ME).
  • Foxp3DTR B6.129(Cg)-Foxp3tm3Ayr /J mice- which express a diphtheria toxin receptor-green fluorescent protein (DTR-GFP) fusion product from an internal ribosome entry site within the Foxp3 3' untranslated region (1)- were a gift from Alexander Rudensky, PhD (Sloan- Kettering Institute, New York, NY). Animals were bred and housed in a pathogen- free facility. All animal protocols were approved by the Johns Hopkins Animal Care and Use Committee. Male mice aged 8-10 weeks were used.
  • DTR-GFP diphtheria toxin receptor-green fluorescent protein
  • DAC 5 -aza-2' -deoxycytidine
  • vehicle containing DMSO 0.38 mL/kg, Invitrogen, Carlsbad, CA
  • DAC 5 -aza-2' -deoxycytidine
  • DMSO vehicle containing DMSO
  • Diphtheria toxin List Biologicals, Campbell, CA
  • PBS 0.1 mL
  • mice Diphtheria toxin-treated WT mice were used as controls for certain of the experiments that used Foxp3DTR mice.
  • Foxp3DTR mice expressed a normal Foxp3 protein and a diphtheria toxin receptor-green fluorescent protein fusion product (DTR-GFP). While mice expressing a Foxp3-GFP fusion protein (Foxp3gfp ) had previously facilitated studies of Treg biology (44), these mice exhibited abnormal Treg epigenetic programming, due to the altered Foxp3 protein (45). Thus, for the current experiments, mice with a normal Foxp3 protein were selected, to ensure fidelity of epigenetic responses.
  • DTR-GFP diphtheria toxin receptor-green fluorescent protein fusion product
  • BAL Bronchoalveolar lavage
  • lungs were excised and prepared for histologic analysis (4). Briefly, lungs were inflated to 25 cm H20 with 1% low-melting agarose (Invitrogen), fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Images were captured at 20x and 200x magnification using a Nikon inverted microscope with Image-Pro Discovery software (Media Cybernetics, Rockville, MD).
  • Erythrocytes were removed using ACK lysis buffer before a second 70- ⁇ nylon strainer filtration and re-suspension in PBS containing 0.5% BSA. Spleen was mashed through a 40- ⁇ nylon strainer, treated with ACK lysis buffer, and re-suspended in 0.5% BSA.
  • Cells were prepared for FACS analysis (4) with fluorochrome-conjugated antibodies purchased from BD Pharmingen, BioLegend (San Diego, CA), and eBioscience (San Diego, CA).
  • Surface stains included PE-Cy7-conjugated anti-CD39, PE-CF594-conjugated anti-CD8 (to confirm exclusion of CD8+ cells from analysis), APC-Cy7-conjugated anti-CD25, Alexa Fluor 700-conjugated anti-CD4, and V500-conjugated anti-CD44.
  • Intracellular stains included PE-, FITC-, or PerCP eFluor 710-conjugated anti-Ki-67, APC-conjugated anti- Foxp3, and BV421-conjugated anti-CTLA-4. Live-dead discrimination was performed with Fixable UV-excitable Blue Dead Cell Stain (Invitrogen). Compensation was completed with UltraComp eBeads (eBioscience). Acquisition was performed using a FACSAria instrument with FACSDiva software (BD) and FlowJo version 7.6.5 (Tree Star, Inc., Ashland, OR) for analysis.
  • Figure 9 shows the Treg gating strategy. Mean fluorescence intensity was calculated as the geometric mean of the positive population fluorescence. Lung cell number was estimated by flow cytometry and confirmed with a hemocytometer using trypan blue exclusion.
  • DNA methylation Tregs cultured with vehicle or 100 nM DAC as above were harvested after 48 hours. DNA was isolated using an AllPrep DNA/RNA Micro kit (Qiagen, Valencia, CA) according to the manufacturer's recommendations. Global DNA methylation was measured on 100 ng of DNA using an Imprint Methylated DNA Quantification kit (Sigma- Aldrich) according to the manufacturer' s recommendations and compared to a methylated DNA control.
  • CD4+ CD25+ cells > 85% Foxp3+
  • CD4+ CD25- cells were isolated using magnetic bead separation (Miltenyi Biotec, Auburn, CA) as previously described (3). Foxp3 positivity using this method was always > 85%.
  • Tregs were then plated in 96-well plates at 2 x 10 5 cells per well containing 200 ⁇ L ⁇ media (5), 1 ⁇ g/mL plate-bound anti-CD3 and soluble anti-CD28 (eBioscience) to provide T cell stimulation, and 40 IU/mL recombinant murine IL-2 (Peprotech, Rocky Hill, NJ) as a survival factor.
  • Cells were then plated in media (28) with plate-bound anti-CD3, soluble anti-CD28 (eBioscience), and recombinant murine IL-2 (Peprotech, Rocky Hill, NJ). Cells were incubated for 48 hours with vehicle or 10 or 100 nM DAC before FACS analysis, DNA methylation measurement, use in suppression assays, or adoptive transfer.
  • Tregs cultured with 100 nM DAC or vehicle as above were incubated with anti-CD3/CD28-coated latex microbeads and CellTrace Violet-pulsed (Invitrogen) CD4+ CD25- effector T cells (Teff) purified by Miltenyi magnetic bead separation (28). Teff did not receive DAC or vehicle treatment. Treg:Teff ratios ranged from 1:8-1 :2 with 1 x 10 5 Teff per well. After 72 hours, effector T cell proliferation was assayed by FACS analysis.
  • mice were used for all experiments and repeated (29). In vitro experiments were performed in triplicate and repeated at least three times. Values are reported as mean + standard error (SEM). Differences between groups were compared using two-tailed Mann- Whitney U tests or Student's t-tests with Holm-Sidak correction for multiple comparisons (mean fluorescence intensity data). Ki-67 positivity was compared using a chi-square test with Yates' correction. Multiple group comparisons were performed using one-way ANOVA or one-way ANOVA on ranks. Mortality differences were analyzed with the Mantel-Cox test. Significance was determined at alpha values less than 0.05.
  • Example 2 DAC Accelerated Recovery from LPS-Induced Acute Lung Injury
  • DAC methyltransferase inhibitor 5-aza-2'-deoxycytidine
  • Lymphocyte-deficient recombinase activating gene- 1 -null (Rag- ⁇ /_ ) mice and Treg depleted (diphtheria toxin-treated Foxp3DTR) mice did not resolve their injury in response to DAC, confirming the criticality of both Rag-1 and Treg to the DAC therapy.
  • Adoptive transfer of only 200,000 DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation, thereby identifying an attractive additional form of therapy (ex vivo cell-based) for lung injury.
  • WT mice received an intratracheal (i.t.) sterile water dose on day 0. They then received daily intraperitoneal (i.p.) DAC or vehicle on days 1 through 4. On day 5 post-water, increased lung Treg frequency was observed in DAC-treated mice, as compared to vehicle-treated mice ( Figure 1A and Figure 13A).
  • Lung Treg Foxp3 expression also increased in the DAC treatment group ( Figure IB and Figure 13B), as determined by FACS analysis using fluorochrome-conjugated antibodies.
  • BAL bronchoalveolar lavage
  • Figure ID Lung histology
  • DAC increased both lung Treg frequency and Foxp3 expression at a non-toxic dose under non-injurious circumstances.
  • DAC increased lung Treg number and Foxp3 expression in a dose that did not cause pulmonary or overt systemic toxicity.
  • Tregs were previously identified to resolve lung injury following LPS administration (3).
  • the cellular basis for the above-identified DAC-induced recovery from lung injury was then examined via assessment of the effect of systemic DAC treatment upon Treg cells post- injury.
  • lung Treg frequency was observed to have increased more than two-fold at day 5 post-injury in response to systemic DAC treatment (Figure 3A), with significant results observed for fixed numbers of cells taken from the right lung ( Figure 3A, left panel), for such cells as a percentage of total cells ( Figure 3A, middle panel) and for such cells as a percentage of CD4 + cells ( Figure 3A, right panel).
  • FIG. 3C shows the Treg phenotypic response to DAC five days after injury.
  • CD44 a Treg activation marker (30)
  • Figure 3C left panel
  • CD39 an ecto-enzyme that catalyzes ATP hydrolysis and serves as an important Treg suppressive mediator
  • Figure 3C middle panel
  • CTLA-4 a powerful negative signal to other immune cells (32), was modestly yet significantly increased (Figure 3C, right panel).
  • DNMT inhibition exerts profound effects on lymphocyte phenotype and function (14), but can affect virtually any cell type.
  • DAC or vehicle was also administered on days 1 through 4 to LPS-injured lymphocyte-deficient (Rag- ⁇ 7 ) mice. Five days post-injury, a time point at which DAC-treated WT mice exhibited a resolving phenotype, Rag- ⁇ 7" mice displayed persistent injury and could not be rescued by DAC.
  • Example 7 Tregs Were Required for DAC-Enhanced Resolution
  • Treg cells were specifically depleted from injured mice, and the effects were examined.
  • Foxp3DTR mice in such mice, diphtheria toxin (DT) administration results in ablation of Treg cells in thymus, lymph nodes, and spleen 2 days after injection, with cell numbers rebounding 10-15 days post-injection) were administered diphtheria toxin beginning two days prior to LPS injury and then every other day thereafter.
  • DT diphtheria toxin
  • DNMT inhibition in culture hypomethylated Tregs and recapitulated some features observed in injured mice (Foxp3 expression, activation, and proliferation) but not increased CD39 expression.
  • DNMT inhibitor-treated Tregs had greater suppressive function in a mixed lymphocyte reaction.
  • DAC-treated mice exhibited a smaller magnitude of weight loss and decreased BAL protein, cell count, neutrophil count, and histologic injury, as compared to vehicle-treated mice ( Figures 8A-8E).
  • DAC-treated animals displayed an increase in lung Treg number and Foxp3 expression ( Figures 8F and 8G).
  • Rescue treatment with DAC exerted favorable effects on lung injury and Tregs in an infectious model of direct lung inflammation.
  • Lung injury and Therapy Intratracheal (i.t.) lipopoly saccharide (LPS, 4-5 mg/kg mouse) was delivered to C57BL/6 (BL/6) WT mice, BALB/c WT mice, and Stat6 'A mice. Mice were treated with systemic rIL-4 complex (rIL-4 2.5 ug/dose + IL-4 antibody (Ab) 15 ug/dose on days 2, 3, +/- 4 or sham) or IL-4 blocking Ab (150 ug/dose on days 1-5 or sham). In addition, Foxp3DTR mice were treated with i.t.
  • systemic rIL-4 complex rIL-4 2.5 ug/dose + IL-4 antibody (Ab) 15 ug/dose on days 2, 3, +/- 4 or sham
  • IL-4 blocking Ab 150 ug/dose on days 1-5 or sham.
  • Foxp3DTR mice were treated with i.t.
  • mice were harvested on day 6 after i.t. LPS for the endpoints described below.
  • IL-4 IL-4 Complex Preparation and Antibody Injections.
  • IL-4 was complexed to an anti-IL-4 antibody to prolong bioavailability, extending the half-life in mice from 0.5 to 24 hours (65).
  • Each dose of IL-4 complex contained 2 ⁇ g of recombinant IL-4 cytokine (PeptroTech) and 15 ⁇ g of an anti-IL-4 antibody (BioXcell, clone 11B 11) suspended in 150 ⁇ of sterile PBS and was administered by i.p. injection on days 2-4 after i.t. LPS, or on days 2-3 after i.t. PAOl vs. sham (150 PBS) unless stated otherwise.
  • anti-IL-4 antibody 200 ⁇ g, BioXcell, clone 11B11 suspended in 150 ⁇ , of sterile PBS or sham (150 ⁇ , PBS) was delivered i.p. on days 1-5 after i.t. LPS.
  • Diphtheria Toxin and Clodronate Liposome Injections were diluted in PBS and administered via i.p. injection on days -2 (50 ⁇ g/kg mouse) and -1 (10 ⁇ g/kg) prior to i.t. LPS, and on days +1, +3, and +5 (10 ⁇ g/kg) following i.t. LPS as previously described (97).
  • Clodronate liposomes (CI 2 MDP) or PBS liposomes (control) were prepared as previously described (100), followed by intravenous (i.v.) instillation after being diluted in 150 ⁇ , of PBS on days 0 and +3 relative to i.t. LPS exposure.
  • mice Male C57BL/6J and BALB/cJ wild type (WT) mice (8-10 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME).
  • Stat6-/- mice on a BALB/c background gift of Dr. Alan Scott
  • Foxp3DTR mice on a C57BL/6 background gift of Dr. Alexander Y. Rudensky of Memorial Sloan-Kettering
  • All mice were housed at the Johns Hopkins University Asthma and Allergy Bldg, and experiments conducted under a protocol approved by the Johns Hopkins Animal Care and Use Committee.
  • mice Preparation of Mice. Animals were anesthetized and the trachea was intubated as previously described (D'Alessio et al. J Clin Invest 119: 2898-2913). Escherichia coli 055:B5 lipopoly saccharide (LPS, 4 mg/kg; Sigma- Aldrich, St. Louis, MO) or sterile water was injected into the trachea. Beginning 24 hours later, rIL-4 or vehicle was administered via daily intraperitoneal (i.p.) injection. Diphtheria toxin (List Biologicals, Campbell, CA) was administered i.p. in a dosing scheme.
  • bronchoalveolar lavage (BAL) fluid analysis and lung histology were performed as previously described (D'Alessio et al. J Clin Invest 119: 2898-2913).
  • Diphtheria toxin-treated WT mice were used as controls for certain of the experiments that used Foxp3DTR mice.
  • Foxp3DTR mice expressed a normal Foxp3 protein and a diphtheria toxin receptor-green fluorescent protein fusion product (DTR- GFP).
  • Intratracheal injections were performed as before (59). Briefly, mice were anesthetized with intraperitoneal (i.p.) ketamine/acetylpromazine (100/2.5 ⁇ g/g) before exposure of the trachea.
  • Lipopolysaccharide (LPS) 3-5 mg/kg mouse weight diluted in sterile water
  • LPS Lipopolysaccharide
  • PAOl Pseudomonas aeruginosa
  • respective vehicle controls were instilled i.t. through a 20-gauge endotracheal catheter on experiment day 0.
  • mice After 4, 5, or 6 days, groups of mice were anesthetized with i.p. ketamine/acetylpromazine and euthanized by exsanguination from the inferior vena cava. The lungs were perfused with 1 ml of phosphate-buffered saline (PBS), followed by
  • BAL bronchoalveolar lavage
  • BAL Analysis BAL was centrifuged at 700 x g for 10 min at 4°C. The cell-free supernatants were stored at -80°C until further analysis. The cell pellet was diluted in PBS, and total cell number was counted with a hemacytometer using trypan blue exclusion. Cell differentials (300 cells per sample) were counted on cytocentrifuge preparation with Diff-Quik stain (Baxter Diagnostics, McGaw Park, IL). Total protein was measured in the cell-free supernatant by the Lowry method (55). Albumin was quantified in the cell-free supernatant by ELISA (Bethyl Laboratories, Montgomery, TX).
  • Lung Histology - H&E Lungs were inflated to a pressure of 25 cmH20 using 1% low melting agarose (Invitrogen, Carlsbad, CA) prior to sectioning and staining with hemotoxylin and eosin (71).
  • Lung Function Measurements On day 6 after i.t. LPS, diffusing capacity was assessed by calculating the diffusion factor for carbon monoxide (DFCO) using a published method (62). In short, the lungs were inflated with 0.8 ml of a gas mixture (-0.5% carbon monoxide (CO), 0.5% neon (Ne), and 99% room air). After a 9 second exposure, the gas mixture was withdrawn from the lungs and diluted to 2 ml with room air. After 15 seconds the recovered gas was injected into a Micro GC gas chromatograph (INFICON, Micro GC Model 3000A, East Syracuse, NY) and the concentrations of Ne and CO were determined.
  • DFCO carbon monoxide
  • the DFCO was defined as 1- (C09/COC)/(Ne9/NeC), where COC and NeC represent the concentration of CO and Ne in the calibration gas and C09 and Ne9 reflect the concentrations of CO and Ne after the 9-second breath hold.
  • mice were paralyzed by administration of 75 mg/kg succinylcholine and connected to a flexiVentTM system (Scireq, Montreal, QC, Canada). Mice were ventilated with 100% oxygen at a tidal volume of 10 ml/kg, a rate of 150 breaths per minute, and a positive end -expiratory pressure (PEEP) of 3 cmH20.
  • PEEP positive end -expiratory pressure
  • RNA and Protein Isolation Whole Lung Homogenate RNA and Protein Isolation. On days 4 and 6 following LPS exposure, lung tissues were homogenized in Trizol Reagent (Life Technologies) and RNA and protein were extracted following phase- separation with chloroform. The aqueous phase was removed and total RNA was precipitated with 100% isopropyl alcohol, washed with 75% ethanol, and redissolved in DEPC-treated water. DNA was removed from the Trizol Reagent (Life Technologies) and RNA and protein were extracted following phase- separation with chloroform. The aqueous phase was removed and total RNA was precipitated with 100% isopropyl alcohol, washed with 75% ethanol, and redissolved in DEPC-treated water. DNA was removed from the Trizol Reagent (Life Technologies) and RNA and protein were extracted following phase- separation with chloroform. The aqueous phase was removed and total RNA was precipitated with 100% isopropyl alcohol, washed with 75% ethanol, and redissolved in DEPC-treated
  • RNA sample concentrations were determined on a NanoDrop 1000 (Thermo Scientific). 1 ⁇ g of total RNA from whole lung homogenates was reverse-transcribed into cDNA with oligo-dT and random primers using an iScript cDNA synthesis kit (Bio-Rad). Gene expression was assessed utilizing TaqMan Gene Expression Assays-On-Demand primer/probe sets and TaqMan Universal Master Mix (Life Technologies) on the Applied Biosystems 7500 real-time PCR system complete with SDS software.
  • 15 ⁇ PCR reactions were performed using 2 ⁇ of cDNA, 0.5 ⁇ of primer/probe set, 7.5 ⁇ of master mix, and 5 ⁇ of DEPC-treated water by initially heating the samples to 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of heating to 95°C for 15 seconds and 60°C for 1 minute.
  • Target gene expression levels were normalized to the housekeeping gene Actinb and the fold change was calculated using the 2 ⁇ ° method.
  • Western Blotting Purified protein concentrations were determined by standard BCA assay (Pierce). SDS-PAGE using 50 ⁇ g of total protein was carried out with the Mini-Protean II System (BioRad) prior to Western blotting.
  • Membranes were blocked in SuperBlock® T20 blocking buffer (Pierce) for 30 minutes at room temperature and then incubated overnight at 4°C with polyclonal rabbit anti-mouse Yml (STEMCELL Technologies), rabbit anti-mouse FIZZ1 (Peprotech), mouse anti-mouse Argl (BD Transduction Laboratories) or rabbit anti-mouse ⁇ - actin (Abeam) antibodies diluted 1: 1500 in blocking solution.
  • lung tissue was minced with scissors into fine pieces, and digested for 30 minutes in a 1 mL solution containing 1 mg/mL collagenase Type II (Invitrogen), 1 mg/mL DNase I (Roche Applied Science), and RPMI 1640 medium (Invitrogen).
  • Lung tissue was ground through a 100 ⁇ cell strainer (BD Biosciences) to form a single-cell suspension, washed, and suspended in ACK buffer (Invitrogen) to lyse red blood cells. The remaining leukocytes were passed through a 100 um cell strainer (BD Biosciences), washed, and suspended in 1 mL of PBS.
  • lxlO 6 cells were stained with the LIVE/DEAD Fixable Blue viability kit according to manufacturer's instructions (Life Technologies), washed, and then incubated in FACS buffer with Fc Block-2.4G2 (BD Pharmingen) antibody to block FCYIII/II receptors for 10 minutes prior to the addition of fluorochrome-conjugated anti-mouse antibodies.
  • isolated cells were first cultured in 1 mL of complete RPMI media containing 5% mouse serum (Jackson Immuno Research) for 4 hours at 37°C with Brefeldin A (eBioscience) and washed twice prior to surface staining. lxlO 6 surface-stained cells were washed, fixed, and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) prior to intracellular staining for 30 minutes with rabbit anti-mouse primary antibodies to FIZZl (1:100, Peprotech).
  • a Cytofix/Cytoperm kit BD Biosciences
  • Intracellular-stained cells were washed once, stained for an additional 30 minutes with a donkey anti-rabbit-BV510 secondary antibody (1:100, Biolegend) for anti-FIZZl, and washed again prior to analysis.
  • Regulatory T cell (Treg) number and activation were assessed by surface staining with anti-CD4-Ax700 (Biolegend) prior to fixation/permeabilization with a FoxP3 Staining Buffer Set
  • IL-4-treated mice exhibited significantly improved survival (92% vs. 57%, Fig. 14A), improved weight gain (Fig. 14B), and reduced histopathologic evidence of lung injury (Fig. 14C) by day 6 after i.t. LPS.
  • Measurement of ALI parameters revealed as much as a three-fold reduction in BAL protein (Fig. 14D), a two-fold reduction in BAL albumin (Fig. 14E), and a five-fold reduction in BAL neutrophils (Fig.
  • mice treated with IL-4 did not alter day 4 BAL protein, albumin, or neutrophil numbers when compared to i.t. H20 plus sham treatment, and both groups had much less lung inflammation compared to LPS-exposed mice.
  • Lung collagen a marker of fibroproliferation, was also significantly reduced in IL-4-treated mice compared to sham at day 6 following i.t. LPS (Fig. 14G).
  • mice exposed to i.t. LPS demonstrated a significant, approximately 25% reduction in Crs and DFCO, as compared to i.t. H20-exposed mice, and approached a DFCO of 0.45, which has been associated with extremely poor gas exchange (similar values were obtained from deceased and exsanguinated mice) (62).
  • Example 13 IL-4 blockade impaired endogenous ALI resolution.
  • FIG. 15C Although the number of alveolar macrophages recovered at day 6 was similar (Fig. 15C), blocking IL-4 reduced the percent and MFI of MMR + or Dectin-1 + (M2 markers) macrophages (Fig. 15D), yet increased CD86 + (Ml marker) macrophages (Fig. 15E).
  • Example 14 Macrophages robustly expressed M2 markers in response to IL-4 and were required to accelerate ALI resolution.
  • IL-4 therapy reduced whole lung mRNA levels of inducible nitric oxide synthase (Nos2), a classical Ml marker, at day 4 (Fig. 16C). Furthermore, mice exposed to i.t.
  • LPS plus IL-4 possessed significantly more FIZZ 1 -expressing monocytes or macrophages within the lung (Fig. 16D).
  • FIZZ 1 -expressing monocytes or macrophages within the lung (Fig. 16D).
  • AM alveolar macrophages
  • IM interstitial macrophages
  • Mo monocytes
  • IL-4 treatment resulted in a significant two-fold increase in the number FIZZl-expressing interstitial macrophages, and a smaller, yet significant increase in FIZZl-expressing alveolar macrophages, as compared to sham.
  • the MFI of FIZZ 1 expression among FIZZ 1 -positive cells was similar. Although a similar number of BAL macrophages were present in sham- and IL-4-treated mice (Fig. 16F), M2 and Ml surface marker expression among alveolar macrophages (F4/80 + ) at day 6 in i.t. LPS- treated mice was also quantified to determine whether IL-4 could sustain macrophage reprogramming 2 days after the last exposure (Fig. 16G). In fact, alveolar macrophages from IL-4-treated mice expressed four- fold higher levels of MMR and Dectin-1, and three-fold lower levels of CD86, as compared to sham treated mice.
  • Example 15 IL-4 therapeutic effects were negated in mice possessing impaired M2 macrophage activation.
  • IL-4-treated WT mice exhibited significant weight gain, as compared to all other groups at days 4 and 5 (Fig. 18A), as well as a greater than two-fold reduction in BAL protein (Fig. 18B) and BAL neutrophils (Fig. 18C) at day 5.
  • Stat6 'A mice Compared to IL-4-treated WT mice, Stat6 'A mice demonstrated impaired resolution with persistently elevated BAL protein and neutrophils, and a blunted response to IL-4. Furthermore, despite a similar number of macrophages recovered by BAL, as compared to WT mice (Fig. 18D), alveolar macrophages from Stat6 'A mice expressed significantly lower levels of MMR and Dectin-1 (Fig. 18E). In contrast to reduced M2 marker expression in Stat6 'A mice, similar CD86 expression between groups (Fig. 18E) was also observed.
  • Tregs CD4 + CD25 + Foxp3 + regulatory T cells
  • IL-4-treated mice demonstrated a significant three-fold increase in Tregs recovered by BAL at day 6 after i.t. LPS (Fig. 19A). Because IL-4 were identified to induce proliferation and maintain full suppressive function of Tregs (87), whether Tregs contributed to the beneficial effects of IL-4 therapy on ALI resolution was examined. Foxp3 DTR mice were treated with i.p. diphtheria toxin to selectively deplete Tregs (Fig. 19B).
  • IL-4 therapy in Foxp3 DTR mice significantly increased weights (Fig. 19C), and significantly reduced parameters of ALI including BAL protein (Fig. 19D), BAL total cell count (Fig. 19E), and BAL neutrophils (Fig. 19F) compared to sham therapy in Foxp3 DTR mice when assessed at day 6 after i.t. LPS.
  • Example 17 IL-4 therapy also accelerated resolution following infection-induced ALI.
  • IL-4-treated mice PAOl on day 0, followed 2 and 3 days later with IL-4 or sham treatment.
  • day 4 with similar lung CFU counts between groups, IL-4- treated mice exhibited significant, two-fold reductions in BAL protein (Fig. 20A) and BAL albumin (Fig. 20B).
  • Fig. 20C BAL neutrophils
  • Fig. 20D IL-4 treatment more than doubled the percentage of alveolar macrophages (F4/80+) that expressed the M2 surface markers MMR and Dectin-1, and also significantly increased the MFI for each marker compared to macrophages from sham-treated mice (Fig. 20E).

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Abstract

La présente invention concerne des procédés et des compositions de traitement de maladies ou de troubles inflammatoires aigus et/ou de traitement d'une lésion pulmonaire. Certains exemples de procédés impliquent l'administration systémique d'un polypeptide d'IL-4 ou d'un fragment de celui-ci possédant une activité cytokine ou d'un agoniste de l'IL-4 à un sujet ayant une maladie ou un trouble inflammatoire et/ou une lésion pulmonaire. D'autres exemples de procédés impliquent l'administration systémique d'un inhibiteur de la méthylation de l'ADN à un sujet et/ou l'administration d'un inhibiteur de la méthylation de l'ADN à une population cellulaire comprenant des lymphocytes T (par exemple, des lymphocytes T reg) pour l'introduction/la re-introduction dans un sujet ayant une maladie ou d'un trouble inflammatoire et/ou une lésion pulmonaire. L'invention concerne également des compositions et des cellules pour leur utilisation dans ces procédés.
PCT/US2016/012621 2015-01-08 2016-01-08 Compositions et procédés d'accélération de la résolution d'une inflammation pulmonaire aiguë WO2016112271A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022020544A1 (fr) * 2020-07-23 2022-01-27 The Board Of Trustees Of The University Of Illinois Méthode de traitement d'une affection inflammatoire
EP4135727A4 (fr) * 2020-04-16 2024-05-22 Univ Pittsburgh Commonwealth Sys Higher Education Vésicules liées à une matrice (mbv) pour le traitement du syndrome de détresse respiratoire aiguë

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115381843B (zh) * 2022-10-28 2023-01-10 四川大学华西医院 L-岩藻糖在制备呼吸系统疾病治疗药物中的用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4522810A (en) 1982-12-09 1985-06-11 The Regents Of The University Of California Feline calicivirus vaccine
US4588580B2 (en) 1984-07-23 1999-02-16 Alaz Corp Transdermal administration of fentanyl and device therefor
CA2721088A1 (fr) 2008-04-11 2009-10-15 University Of Southern California Procedes et compositions pour accelerer la generation de cellules t regulatrices ex vivo
KR101535319B1 (ko) 2008-11-05 2015-07-10 유니버시티 오브 써던 캘리포니아 후생적 조절의 소형 분자 조절물질, 그리고 이들의 치료적 적용
WO2013050596A1 (fr) 2011-10-06 2013-04-11 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé pour conserver l'expression de foxp3 dans le lymphocyte t régulateur après expansion

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHAN, M. W. Y. ET AL.: "Low-dose 5-aza-2' -deoxycytidine pretreatment inhibits experimental autoimmune encephalomyelitis by induction of regulatory T cells", MOLECULAR MEDICINE, vol. 20, 2014, pages 248 - 256 *
LEE, S. W. ET AL.: "Anti-inflammatory effects of IL -4 and IL -10 on human polymorphonuclear leukocytes", JOURNAL OF KOREAN MEDICAL SCIENCE, vol. 17, 2002, pages 7 - 14 *
MEDURI, G. U. ET AL.: "Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS: plasma IL -1beta and IL -6 levels are consistent and efficient predictors of outcome over time", CHEST JOURNAL, vol. 107, no. 4, 1995, pages 1062 - 1073 *
QUDDUS, J. ET AL.: "Treating activated CD 4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice", JOURNAL OF CLINICAL INVESTIGATION, vol. 92, 1993, pages 38 - 53 *
SINGER, B. D. ET AL.: "Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation", AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, vol. 52, no. 5, 8 October 2014 (2014-10-08), pages 641 - 652 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4135727A4 (fr) * 2020-04-16 2024-05-22 Univ Pittsburgh Commonwealth Sys Higher Education Vésicules liées à une matrice (mbv) pour le traitement du syndrome de détresse respiratoire aiguë
WO2022020544A1 (fr) * 2020-07-23 2022-01-27 The Board Of Trustees Of The University Of Illinois Méthode de traitement d'une affection inflammatoire

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